Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly

Similar to other positive-sense, single-stranded RNA viruses, hepatitis C virus (HCV) replicates its genome in a remodeled intracellular membranous structure known as the membranous web (MW). To date, the process of MW formation remains unclear. It is generally acknowledged that HCV nonstructural protein 4B (NS4B) can induce MW formation through interaction with the cytosolic endoplasmic reticulum (ER) membrane. Many host proteins, such as phosphatidylinositol 4-kinase IIIα (PI4KIIIα), have been identified as critical factors required for this process. We now report a new factor, the cytosolic phospholipase A2 gamma (PLA2G4C), which contributes to MW formation, HCV replication, and assembly. The PLA2G4C gene was identified as a host gene with upregulated expression upon HCV infection. Knockdown of PLA2G4C in HCV-infected cells or HCV replicon-containing cells by small interfering RNA (siRNA) significantly suppressed HCV replication and assembly. In addition, the chemical inhibitor methyl arachidonyl fluorophosphonate (MAFP), which specifically inhibits PLA2, reduced HCV replication and assembly. Electron microscopy demonstrated that MW structure formation was defective after PLA2G4C knockdown in HCV replicon-containing cells. Further analysis by immunostaining and immunoprecipitation assays indicated that PLA2G4C colocalized with the HCV proteins NS4B and NS5A in cells infected with JFH-1 and interacted with NS4B. In addition, PLA2G4C was able to transport the HCV nonstructural proteins from replication sites to lipid droplets, the site for HCV assembly. These data suggest that PLA2G4C plays an important role in the HCV life cycle and might represent a potential target for anti-HCV therapy.     

Novel C5a Agonist-Based Dendritic Cell Vaccine in a Murine Model of Melanoma

A novel method to improve targeting and presentation of poorly immunogenic tumor-related antigens was investigated. This was performed with a molecular adjuvant constructed by covalently linking a response selective peptide agonist of C5a (YSFKDMP(MeL)aR) to known melanoma tumor-related antigens. C57Bl/6J mice were injected subcutaneously with bone marrow derived dendritic cells (DCs) pulsed with a melanoma epitope (TRP2-P2/Agonist), melanoma epitope tyrosinase (TYR/Agonist), a nonfunctional reverse conformation C5a agonist bound to TYR(reverse peptide) or DMSO-PBS vehicle. Mice were injected with the pulsed DCs and cytokines IL-2 and GM-CSF three times prior to subcutaneous challenge with B16-F10 melanoma cells. All groups subsequently received DC vaccine boosters twice per week. Tumor growth was reduced and survival enhanced in mice immunized with the combination of TRP2-P2/Agonist and TYR/Agonist compared to mice receiving reverse peptide or vehicle.     

Dendritic Signals Command Firing Dynamics in a Mathematical Model of Cerebellar Purkinje Cells

Dendrites of cerebellar Purkinje cells (PCs) respond to brief excitations from parallel fibers with lasting plateau depolarizations. It is unknown whether these plateaus are local events that boost the synaptic signals or they propagate to the soma and directly take part in setting the cell firing dynamics. To address this issue, we analyzed a likely mechanism underlying plateaus in three representations of a reconstructed PC with increasing complexity. Analysis in an infinite cable suggests that Ca plateaus triggered by direct excitatory inputs from parallel fibers and their mirror signals, valleys (putatively triggered by the local feed forward inhibitory network), cannot propagate. However, simulations of the model in electrotonic equivalent cables prove that Ca plateaus (resp. valleys) are conducted over the entire cell with velocities typical of passive events once they are triggered by threshold synaptic inputs that turn the membrane current inward (resp. outward) over the whole cell surface. Bifurcation analysis of the model in equivalent cables, and simulations in a fully reconstructed PC both indicate that dendritic Ca plateaus and valleys, respectively, command epochs of firing and silencing of PCs.     

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction^{1, 2}. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells^{1, 2}. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells^{1, 2}. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide^{1, 2}. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.Download video file.^{(152M, mp4)}     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

Spontaneous Autoimmunity in the Absence of IL-2 Is Driven by Uncontrolled Dendritic Cells

BALB/c IL-2-deficient (IL-2-KO) mice develop systemic autoimmunity, dying within 3 to 5 wk from complications of autoimmune hemolytic anemia. Disease in these mice is Th1 mediated, and IFN-γ production is required for early autoimmunity. In this study, we show that dendritic cells (DCs) are required for optimal IFN-γ production by T cells in the IL-2-KO mouse. Disease is marked by DC accumulation, activation, and elevated production of Th1-inducing cytokines. IL-2-KO DCs induce heightened proliferation and cytokine production by naive T cells compared with wild-type DCs. The depletion of either conventional or plasmacytoid DCs significantly prolongs the survival of IL-2-KO mice, demonstrating that DCs contribute to the progression of autoimmunity. Elimination of Th1-inducing cytokine signals (type 1 IFN and IL-12) reduces RBC-specific Ab production and augments survival, indicating that cytokines derived from both plasmacytoid DCs and conventional DCs contribute to disease severity. DC activation likely precedes T cell activation because DCs are functionally activated even in an environment lacking overt T cell activation. These data indicate that both conventional and plasmacytoid DCs are critical regulators in the development of this systemic Ab-mediated autoimmune disease, in large part through the production of IL-12 and type 1 IFNs.     

An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis

Objective

The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.

Methods

In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.

Results

Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1β and IL-6, and of chemokines like MIP-1α and MCP-1.

Conclusion

The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.     

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.     

US28 Is a Potent Activator of Phospholipase C during HCMV Infection of Clinically Relevant Target Cells

Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca⁺⁺ release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca⁺⁺ release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.     

p21 Is a Critical CDK2 Regulator Essential for Proliferation Control in Rb-deficient Cells

Proliferation in mammalian cells is controlled primarily in the G1-phase of the cell cycle through the action of the G1 cyclin–dependent kinases, CDK4 and CDK2. To explore the mechanism of cellular response to extrinsic factors, specific loss of function mutations were generated in two negative regulators of G1 progression, p21 and pRB. Individually, these mutations were shown to have significant effects in G1 regulation, and when combined, Rb and p21 mutations caused more profound defects in G1. Moreover, cells deficient for pRB and p21 were uniquely capable of anchorage-independent growth. In contrast, combined absence of pRB and p21 function was not sufficient to overcome contact inhibition of growth nor for tumor formation in nude mice. Finally, animals with the genotype Rb^+/−;p21^−/− succumbed to tumors more rapidly than Rb^+/− mice, suggesting that in certain contexts mutations in these two cell cycle regulators can cooperate in tumor development.     

Mitochondrial Dynamics Is Critical for the Full Pluripotency and Embryonic Developmental Potential of Pluripotent Stem Cells

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EBI2 Is a Negative Regulator of Type I Interferons in Plasmacytoid and Myeloid Dendritic Cells

Epstein-Barr virus induced receptor 2 (EBI2), a Gα_i-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b⁺ myeloid cells. Activation of EBI2^−/− pDCs and CD11b⁺ cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, in vivo challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2^−/− mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b⁺ cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.     

CXCL10 Is Critical for the Generation of Protective CD8 T Cell Response Induced by Antigen Pulsed CpG-ODN Activated Dendritic Cells

The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8⁺ T cell-mediated immune response. We observed significant reduction of CD8⁺ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8⁺ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8⁺ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against Leishmania donovani infection.     

Evidence for a Critical Role of Catecholamines for Cardiomyocyte Lineage Commitment in Murine Embryonic Stem Cells

Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and β-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and β-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.     

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer''s patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.     

SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.     

Phage Therapy Is Effective against Infection by Mycobacterium ulcerans in a Murine Footpad Model

Background

Buruli Ulcer (BU) is a neglected, necrotizing skin disease caused by Mycobacterium ulcerans. Currently, there is no vaccine against M. ulcerans infection. Although the World Health Organization recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical management of advanced stages is still based on the surgical resection of infected skin. The use of bacteriophages for the control of bacterial infections has been considered as an alternative or to be used in association with antibiotherapy. Additionally, the mycobacteriophage D29 has previously been shown to display lytic activity against M. ulcerans isolates.

Methodology/Principal findings

We used the mouse footpad model of M. ulcerans infection to evaluate the therapeutic efficacy of treatment with mycobacteriophage D29. Analyses of macroscopic lesions, bacterial burdens, histology and cytokine production were performed in both M. ulcerans-infected footpads and draining lymph nodes (DLN). We have demonstrated that a single subcutaneous injection of the mycobacteriophage D29, administered 33 days after bacterial challenge, was sufficient to decrease pathology and to prevent ulceration. This protection resulted in a significant reduction of M. ulcerans numbers accompanied by an increase of cytokine levels (including IFN-γ), both in footpads and DLN. Additionally, mycobacteriophage D29 treatment induced a cellular infiltrate of a lymphocytic/macrophagic profile.

Conclusions/Significance

Our observations demonstrate the potential of phage therapy against M. ulcerans infection, paving the way for future studies aiming at the development of novel phage-related therapeutic approaches against BU.     

SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.     

Up-Regulation of GITRL on Dendritic Cells by WGP Improves Anti-Tumor Immunity in Murine Lewis Lung Carcinoma

Background

β-Glucans have been shown to function as a potent immunomodulator to stimulate innate and adaptive immune responses, which contributes to their anti-tumor property. However, their mechanisms of action are still elusive. Glucocorticoid-induced TNF receptor ligand (GITRL), a member of the TNF superfamily, binds to its receptor, GITR, on both effector and regulatory T cells, generates a positive co-stimulatory signal implicated in a wide range of T cell functions, which is important for the development of immune responses.

Methodology/Principal Findings

In this study, we found that whole β-glucan particles (WGPs) could activate dendritic cells (DCs) via dectin-1 receptor, and increase the expression of GITRL on DCs in vitro and in vivo. Furthermore, we demonstrated that the increased GITRL on DCs could impair the regulartory T cell (Treg)-mediated suppression and enhance effector T cell proliferation in a GITR/GITRL dependent way. In tumor models, DCs with high levels of GITRL were of great potential to prime cytotoxic T lymphocyte (CTL) responses and down-regulate the suppressive activity of Treg cells, thereby leading to the delayed tumor progression.

Conclusions/Significance

These findings suggest that particulate β-glucans can be used as an immunomodulator to stimulate potent T cell-mediated adaptive immunity while down-regulate suppressive immune activity via GITR/GITRL interaction, leading to a more efficient defense mechanism against tumor development.     

Reproduction and Growth in a Murine Model of Early Life-Onset Inflammatory Bowel Disease

Studies in transgenic murine models have provided insight into the complexity underlying inflammatory bowel disease (IBD), a disease hypothesized to result from an injurious immune response against intestinal microbiota. We recently developed a mouse model of IBD that phenotypically and histologically resembles human childhood-onset ulcerative colitis (UC), using mice that are genetically modified to be deficient in the cytokines TNF and IL-10 (“T/I” mice). Here we report the effects of early life onset of colon inflammation on growth and reproductive performance of T/I mice. T/I dams with colitis often failed to get pregnant or had small litters with pups that failed to thrive. Production was optimized by breeding double homozygous mutant T/I males to females homozygous mutant for TNF deficiency and heterozygous for deficiency of IL-10 (“T/I-het” dams) that were not susceptible to spontaneous colon inflammation. When born to healthy (T/I-het) dams, T/I pups initially gained weight similarly to wild type (WT) pups and to their non-colitis-susceptible T/I-het littermates. However, their growth curves diverged between 8 and 13 weeks, when most T/I mice had developed moderate to severe colitis. The observed growth failure in T/I mice occurred despite a significant increase in their food consumption and in the absence of protein loss in the stool. This was not due to TNF-induced anorexia or altered food consumption due to elevated leptin levels. Metabolic studies demonstrated increased consumption of oxygen and water and increased production of heat and CO₂ in T/I mice compared to their T/I-het littermates, without differences in motor activity. Based on the clinical similarities of this early life onset model of IBD in T/I mice to human IBD, these results suggest that mechanisms previously hypothesized to explain growth failure in children with IBD require re-evaluation. The T/I mouse model may be useful for further investigation of such mechanisms and for development of therapies to prevent reproductive complications and/or growth failure in humans with IBD.