bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunomodulation via Novel Use of TLR4 by the Filarial Nematode Phosphorylcholine-Containing Secreted Product, ES-62
Authors: Goodridge Helen S. , Marshall Fraser A. , Else Kathryn J. , Houston Katrina M. , Egan Caitlin , Al-Riyami Lamyaa , Liew Foo-Yew , Harnett William , Harnett Margaret M.
Published: April 2014

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-α, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-γ. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-α production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.
IL-12, but Not IFN-α, Promotes STAT4 Activation and Th1 Development in Murine CD4+ T Cells Expressing a Chimeric Murine/Human Stat2 Gene
Authors: Persky Meredith E. , Murphy Kenneth M. , Farrar J. David
Published: April 2014

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4+ Th1 development in both murine and human T cells, IFN-αβ drives Th1 development only in human cells. This IFN-αβ-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4+ T cell responses to IFN-αβ. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4+ T cells and was activated by murine IFN-α signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-α-driven Th1 development as defined by IFN-γ expression. Furthermore, IL-12, but not IFN-α, promoted acute IFN-γ secretion in collaboration with IL-18 stimulation in both CD4+ and CD8+ T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-α. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-αβR), it is not sufficient to restore this process through the murine IFNAR complex.
Vα and Vβ Public Repertoires Are Highly Conserved in Terminal Deoxynucleotidyl Transferase-Deficient Mice
Authors: Fazilleau Nicolas , Cabaniols Jean-Pierre , Lemaître Fabrice , Motta Iris , Kourilsky Philippe , Kanellopoulos Jean M.
Published: April 2014

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Vα-Jα and Vβ-Dβ-Jβ rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the αβ TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT°/° mice, even for the CDR3 loops which are shorter than those found in TdT+/+ animals. Moreover, the 10- to 20-fold decrease in αβ T cell diversity in TdT°/° mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3β in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3β. We interpret our findings as suggesting that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHCα helices are involved in the selection of public repertoires.
Cutting Edge: Epitope-Dependent Effect of Nef-Mediated HLA Class I Down-Regulation on Ability of HIV-1-Specific CTLs to Suppress HIV-1 Replication
Authors: Tomiyama Hiroko , Fujiwara Mamoru , Oka Shinichi , Takiguchi Masafumi
Published: April 2014

It is believed that Nef-mediated HLA class I down-regulation is one of the mechanisms that allow HIV-1-infected cells to escape from being killed by HIV-1-specific human CTLs. In this study, we show that the effect of Nef-mediated HLA class I down-regulation on the ability of HIV-1-specific CTLs to suppress HIV-1 replication is epitope dependent. The CTLs specific for two Pol epitopes presented by HLA-B*5101, one of the HLA alleles associated with slow progression to AIDS, effectively killed HIV-1-infected CD4+ T cells and suppressed HIV-1 replication. In contrast, those specific for the other four epitopes failed to kill HIV-1-infected CD4+ T cells and partially or hardly suppressed HIV-1 replication. The difference of the ability between these two types of CTLs may result from the difference of the number of HLA class I epitope complex on the surface of NL-432-infected CD4+ T cells.
Expression of Cytokine, Chemokine, and Adhesion Molecules during Endothelial Cell Activation Induced by Antibodies against Dengue Virus Nonstructural Protein 1
Authors: Lin Chiou-Feng , Chiu Shu-Chen , Hsiao Yu-Ling , Wan Shu-Wen , Lei Huan-Yao , Shiau Ai-Li , Liu Hsiao-Sheng , Yeh Trai-Ming , Chen Shun-Hua , Liu Ching-Chuan , Lin Yee-Shin
Published: April 2014

Vascular dysfunction is a hallmark associated with disease onset in dengue hemorrhagic fever and dengue shock syndrome. In addition to direct viral damage, immune responses to dengue virus (DV) infection may also underlie the pathogenesis of disease. We have proposed a mechanism of molecular mimicry in which Abs directed against DV nonstructural protein 1 (NS1) cross-react with endothelial cells and induce damage. In this study, we demonstrated the inflammatory endothelial cell activation induced by anti-DV NS1 via the transcription factor NF-κB-regulated pathway. Protein phosphorylation and NF-κB activation were observed after anti-DV NS1 stimulation in a human microvascular endothelial cell line-1. The cytokine and chemokine production, including IL-6, IL-8, and MCP-1, but not RANTES, in endothelial cells increased after treatment with anti-DV NS1 Abs. The expression of IL-6, IL-8, and MCP-1 was blocked by the preabsorption of anti-DV NS1 with DV NS1 or by the inhibition of NF-κB activation. Furthermore, the increases in both ICAM-1 expression and the ability of human PBMC to adhere to endothelial cells were also observed, and these effects were inhibited by pretreatment with anti-ICAM-1 or anti-MCP-1 Abs. Therefore, in addition to endothelial cell apoptosis, as previously reported, inflammatory activation occurs in endothelial cells after stimulation by anti-DV NS1 Abs. These results suggest the involvement of anti-DV NS1 Abs in the vasculopathy of DV infection.
Enhancement of Dendritic Cell Production by Fms-Like Tyrosine Kinase-3 Ligand Increases the Resistance of Mice to a Burn Wound Infection
Authors: Toliver-Kinsky Tracy E. , Cui Weihua , Murphey Erle D. , Lin Chengyie , Sherwood Edward R.
Published: April 2014

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.
Modulation of Proinflammatory Responses to Pneumocystis carinii f. sp. muris in Neonatal Mice by Granulocyte-Macrophage Colony-Stimulating Factor and IL-4: Role of APCs
Authors: Qureshi Mahboob H. , Empey Kerry M. , Garvy Beth A.
Published: April 2014

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c+ dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-α, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c+ DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4+CD44highCD62Llow cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.
Differential Immunogenicity of Various Heterologous Prime-Boost Vaccine Regimens Using DNA and Viral Vectors in Healthy Volunteers
Authors: Vuola Jenni M. , Keating Sheila , Webster Daniel P. , Berthoud Tamara , Dunachie Susanna , Gilbert Sarah C. , Hill Adrian V. S.
Published: April 2014

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-γ response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-γ response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-γ responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-γ response was mainly due to CD4+ T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8+ T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.
Hemozoin Induces Macrophage Chemokine Expression through Oxidative Stress-Dependent and -Independent Mechanisms
Authors: Jaramillo Maritza , Godbout Marianne , Olivier Martin
Published: April 2014

Chemokine production has been associated with the immunopathology related to malaria. Previous findings indicated that hemozoin (HZ), a parasite metabolite released during schizogeny, might be an important source of these proinflammatory mediators. In this study we investigated the molecular mechanisms underlying HZ-inducible macrophage (Mφ) chemokine mRNA expression. We found that both Plasmodium falciparum HZ and synthetic HZ increase mRNA levels of various chemokine transcripts (MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2) in murine B10R Mφ. The cellular response to HZ involved ERK1/2 phosphorylation, NF-κB activation, reactive oxygen species (ROS) generation, and ROS-dependent protein-tyrosine phosphatase down-regulation. Selective inhibition of either IκBα or the ERK1/2 pathway abolished both NF-κB activation and chemokine up-regulation. Similarly, blockage of HZ-inducible Mφ ROS with superoxide dismutase suppressed chemokine induction, strongly reduced NF-κB activation, and restored HZ-mediated Mφ protein-tyrosine phosphatase inactivation. In contrast, superoxide dismutase had no effect on EKR1/2 phosphorylation by HZ. Collectively, these data indicate that HZ triggers ROS-dependent and -independent signals, leading to increased chemokine mRNA expression in Mφ. Overall, our findings may help to better understand the molecular mechanisms through which parasite components, such as HZ, modulate the immune response during malaria infection.
TNF-α Is a Critical Effector and a Target for Therapy in Antiphospholipid Antibody-Induced Pregnancy Loss
Authors: Berman Jessica , Girardi Guillermina , Salmon Jane E.
Published: April 2014

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, intrauterine growth restriction, and vascular thrombosis in the presence of antiphospholipid (aPL) Abs. Our studies in a murine model of APS induced by passive transfer of human aPL Abs have shown that activation of complement and recruitment of neutrophils into decidua are required for fetal loss, and emphasize the importance of inflammation in aPL Ab-induced pregnancy loss. In this study, we examine the role of TNF-α in pregnancy complications associated with aPL Abs in a murine model of APS. We show that aPL Abs are specifically targeted to decidual tissue and cause a rapid increase in decidual and systemic TNF-α levels. We identify the release of TNF-α as a critical intermediate that acts downstream of C5 activation, based on the fetal protective effects of TNF-α deficiency and TNF blockade and on the absence of increased TNF-α levels in C5-deficient mice treated with aPL Abs. Our results suggest that TNF-α links pathogenic aPL Abs to fetal damage and identify TNF blockade as a potential therapy for the pregnancy complications of APS.
SHIP Down-Regulates FcεR1-Induced Degranulation at Supraoptimal IgE or Antigen Levels
Authors: Gimborn Kerstin , Lessmann Eva , Kuppig Stephan , Krystal Gerald , Huber Michael
Published: April 2014

Cross-linking of the IgE-loaded high-affinity IgE receptor (FcεR1) by multivalent Ags results in mast cell activation and subsequent release of multiple proinflammatory mediators. The dose-response curve for FcεR1-mediated degranulation is bell-shaped, regardless of whether the IgE or the Ag concentration is varied. Although overall calcium influx follows this bell-shaped curve, intracellular calcium release continues to increase at supraoptimal IgE or Ag concentrations. As well, overall calcium mobilization adopts more transient kinetics when stimulations are conducted with supraoptimal instead of optimal Ag concentrations. Moreover, certain early signaling events continue to increase whereas degranulation drops under supraoptimal conditions. We identified SHIP, possibly in association with the FcεR1 β-chain, as a critical negative regulator acting within the inhibitory (supraoptimal) region of the dose-response curve that shifts the kinetics of calcium mobilization from a sustained to a transient response. Consistent with this, we found that degranulation of SHIP-deficient murine bone marrow-derived mast cells was not significantly reduced at supraoptimal Ag levels. A potential mediator of SHIP action, Bruton’s tyrosine kinase, did not seem to play a role within the supraoptimal suppression of degranulation. Interestingly, SHIP was found to colocalize with the actin cytoskeleton (which has been shown previously to mediate the inhibition of degranulation at supraoptimal Ag doses). These results suggest that SHIP, together with other negative regulators, restrains bone marrow-derived mast cell activation at supraoptimal IgE or Ag concentrations in concert with the actin cytoskeleton.
Stimulation of Host NKT Cells by Synthetic Glycolipid Regulates Acute Graft-versus-Host Disease by Inducing Th2 Polarization of Donor T Cells
Authors: Hashimoto Daigo , Asakura Shoji , Miyake Sachiko , Yamamura Takashi , Van Kaer Luc , Liu Chen , Tanimoto Mitsune , Teshima Takanori
Published: April 2014

NKT cells are a unique immunoregulatory T cell population that produces large amounts of cytokines. We have investigated whether stimulation of host NKT cells could modulate acute graft-vs-host disease (GVHD) in mice. Injection of the synthetic NKT cell ligand α-galactosylceramide (α-GalCer) to recipient mice on day 0 following allogeneic bone marrow transplantation promoted Th2 polarization of donor T cells and a dramatic reduction of serum TNF-α, a critical mediator of GVHD. A single injection of α-GalCer to recipient mice significantly reduced morbidity and mortality of GVHD. However, the same treatment was unable to confer protection against GVHD in NKT cell-deficient CD1d knockout (CD1d−/−) or IL-4−/− recipient mice or when STAT6−/− mice were used as donors, indicating the critical role of host NKT cells, host production of IL-4, and Th2 cytokine responses mediated by donor T cells on the protective effects of α-GalCer against GVHD. Thus, stimulation of host NKT cells through administration of NKT ligand can regulate acute GVHD by inducing Th2 polarization of donor T cells via STAT6-dependent mechanisms and might represent a novel strategy for prevention of acute GVHD.
Dendritic Cells Loaded with Stressed Tumor Cells Elicit Long-Lasting Protective Tumor Immunity in Mice Depleted of CD4+CD25+ Regulatory T Cells
Authors: Prasad Simon J. , Farrand Kathryn J. , Matthews Stephanie A. , Chang Joe H. , McHugh Rebecca S. , Ronchese Franca
Published: April 2014

Dendritic cell (DC)-based vaccination represents a promising approach to harness the specificity and potency of the immune system to combat cancer. Finding optimal strategies for tumor Ag preparation and subsequent pulsing of DC, as well as improving the immunogenicity of weak tumor Ags remain among the first challenges of this approach. In this report, we use a prophylactic vaccine consisting of DC loaded with whole, nonmanipulated B16-F10 melanoma cells that had been stressed by heat shock and gamma irradiation. Stressed B16-F10 cells underwent apoptosis and were internalized by bone marrow-derived DC during coculture. Surprisingly, coculture of DC with stressed B16-F10 undergoing apoptosis and necrosis did not induce DC maturation. However, a marked retardation in tumor growth was observed in C57BL/6 mice immunized using DC loaded with stressed B16-F10 cells and subsequently challenged with B16-F10 cells. Growth retardation was further increased by treating DC with LPS before in vivo administration. In vivo depletion studies revealed that both CD8+ and CD4+ T cells played a critical role in retarding tumor growth. In addition, treatment with anti-CD25 Ab to deplete CD4+CD25+ regulatory T cells before DC vaccination considerably improved the effect of the vaccine and allowed the development of long-lived immune responses that were tumor protective. Our results demonstrate that depletion of regulatory T cells is an effective approach to improving the success of DC-based vaccination against weakly immunogenic tumors. Such a strategy can be readily applied to other tumor models and extended to therapeutic vaccination settings.
IFN-αβ Secreted during Infection Is Necessary but Not Sufficient for Negative Feedback Regulation of IFN-αβ Signaling byMycobacterium tuberculosis
Authors: Prabhakar Savita , Qiao Yaming , Canova Antony , Tse Doris B. , Pine Richard
Published: April 2014

IFN-αβ functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-αβ secretion and down-regulation of IFN-αβ signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-αβ prevents inhibition of IFN-α signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-αβ. First, greater inhibition of IFN-α-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-αβ pretreatment. Second, LPS also induces IFN-αβ secretion and causes IFN-αβ-dependent down-regulation of IFN-α signaling, yet the inhibition differs from that caused by infection. Third, IFN-α signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-αβ is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-αβ receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-α stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-α signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.
Distinct Roles for IL-4 and IL-10 in Regulating T2 Immunity during Allergic Bronchopulmonary Mycosis
Authors: Hernandez Yadira , Arora Shikha , Erb-Downward John R. , McDonald Roderick A. , Toews Galen B. , Huffnagle Gary B.
Published: April 2014

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-γ levels remained the same. TNF-α and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.
CXC Chemokine Ligand 16 Promotes Integrin-Mediated Adhesion of Liver-Infiltrating Lymphocytes to Cholangiocytes and Hepatocytes within the Inflamed Human Liver
Authors: Heydtmann Mathis , Lalor Patricia F. , Eksteen J. Albertus , Hübscher Stefan G. , Briskin Mike , Adams David H.
Published: April 2014

Lymphocyte recruitment to the liver is critical for viral clearance in acute hepatitis and in the pathogenesis of chronic inflammatory liver disease when persistent chronic inflammation leads to fibrosis and cirrhosis. Chemokines regulate leukocyte recruitment and positioning in tissues and are thus critical regulators of chronic inflammation. The chemokine CXCL16, which is found in liver tissue, exists in a transmembrane as well as soluble form, providing a potential mechanism for localization to particular structures. We studied the role of CXCL16 and its receptor CXCR6 in lymphocyte recruitment and retention in the liver. A higher proportion of CXCR6+ T cells was detected in blood of hepatitis C virus patients compared with healthy subjects, and in chronic inflammatory liver disease >60% of intrahepatic T cells expressed CXCR6, including CD4, CD8, and CD56+ T cells compared with <30% in matched blood samples. CXCR6+ lymphocytes were found in association with CXCL16+ bile ducts in portal tracts and with hepatocytes at sites of interface hepatitis. Analysis of CXCL16 expression and subcellular distribution in cultured human cholangiocytes, sinusoidal endothelial cells, and hepatocytes revealed that all three cell types expressed CXCL16, with the strongest staining seen on cholangiocytes. CXCL16 on the cholangiocyte membrane was able to support lymphocyte adhesion by triggering conformational activation of β1 integrins and binding to VCAM-1. Thus, CXCL16 can promote lymphocyte adhesion to epithelial cells and may function to attract and retain effector cells that promote biliary and hepatocyte destruction in inflammatory liver disease.
Phospholipids Inhibit Lipopolysaccharide (LPS)-Induced Cell Activation: A Role for LPS-Binding Protein
Authors: Mueller Mareile , Brandenburg Klaus , Dedrick Russ , Schromm Andra B. , Seydel Ulrich
Published: April 2014

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-α release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.
Cutting Edge: Species-Specific TLR9-Mediated Recognition of CpG and Non-CpG Phosphorothioate-Modified Oligonucleotides
Authors: Roberts Tara L. , Sweet Matthew J. , Hume David A. , Stacey Katryn J.
Published: April 2014

Different DNA motifs are required for optimal stimulation of mouse and human immune cells by CpG oligodeoxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CpG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 MAPK than optimal motifs. When the CpG dinucleotide was inverted to GC in each ODN, some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may be responsible for mediating many published CpG-independent responses to PS-ODN.
Cutting Edge: Role of STAT1, STAT3, and STAT5 in IFN-αβ Responses in T Lymphocytes
Authors: Tanabe Yoshinari , Nishibori Takeaki , Su Leon , Arduini Robert M. , Baker Darren P. , David Michael
Published: April 2014

Engagement of the IFN-αβ receptor initiates multiple signaling cascades, including activation of the STAT. In this study, we demonstrate that IFN-αβ, although antiproliferative in wild-type CD4+ or CD8+ T cells, act as strong mitogens on their STAT1−/− counterparts. Furthermore, IFN-αβ exert little effect on apoptosis in wild-type cells, but are potent survival factors in the absence of STAT1. The antiapoptotic response in the absence of STAT1 is predominantly mediated by STAT3, and to a lesser extent by STAT5A/B. In contrast, the mitogenic IFN-αβ response gained through the absence of STAT1 is only marginally affected when STAT5A/B expression is also abrogated, but is completely dependent on STAT3 activation. These findings provide the first evidence for a function of STAT3 and STAT5A/B in the IFN-αβ response, and support a model in which the IFN-αβ receptor initiates both pro- and antiapoptotic responses through STAT1, and STAT3 and STAT5A/B, respectively.
Cutting Edge: MHC Class II-Restricted Killing In Vivo during Viral Infection
Authors: Jellison Evan R. , Kim Sung-Kwon , Welsh Raymond M.
Published: April 2014

Class II-restricted CD4 T cell-mediated killing of target cells has previously been documented in vitro but not in vivo. In this study, we demonstrate CD4-dependent MHC class II-restricted killing in lymphocytic choriomeningitis virus-infected mice in vivo using an in vivo cytotoxicity assay that features class II-expressing B cells as targets.
Peripheral “CD8 Tuning” Dynamically Modulates the Size and Responsiveness of an Antigen-Specific T Cell Pool In Vivo
Authors: Maile Robert , Siler Catherine A. , Kerry Samantha E. , Midkiff Katherine E. , Collins Edward J. , Frelinger Jeffrey A.
Published: April 2014

In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8+ T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8low T cells produced after peripheral HY-Db MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8int T cells from heterozygote CD8+/0 mice. Finally, we used pre-existing nonresponsive CD8low T cells from male HY TCR transgenic mice. In CD8low and CD8high mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (KD) and dissociation constant (T1/2) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8high T cells of the same specificity. Additionally, direct injections of wild-type HY-Db and C2A-Db tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8low T cells, yet C2A-Db was superior in inducing a primed CD8+CD44+ memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by “CD8 tuning” of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.
Heterogeneity of Expression of IgA Receptors by Human, Mouse, and Rat Eosinophils
Authors: Decot Véronique , Woerly Gaëtane , Loyens Marc , Loiseau Sylvie , Quatannens Brigitte , Capron Monique , Dombrowicz David
Published: April 2014

IgA is the most abundant class of Abs at mucosal surfaces where eosinophils carry out many of their effector functions. Most of the known IgA-mediated functions require interactions with IgA receptors, six of which have been identified in humans. These include the IgA FcR FcαRI/CD89 and the receptor for the secretory component, already identified on human eosinophils, the polymeric IgR, the Fcα/μR, asialoglycoprotein (ASGP)-R, and transferrin (Tf)R/CD71. In rodents, the existence of IgA receptors on mouse and rat eosinophils remains unclear. We have compared the expression and function of IgA receptors by human, rat, and mouse eosinophils. Our results show that human eosinophils express functional polymeric IgR, ASGP-R, and TfR, in addition to CD89 and the receptor for the secretory component, and that IgA receptors are expressed by rodent eosinophils. Indeed, mouse eosinophils expressed only TfR, whereas rat eosinophils expressed ASGP-R and CD89 mRNA. These results provide a molecular basis for the differences observed between human, rat, and mouse regarding IgA-mediated immunity.
Differential Activation of Anti-Erythrocyte and Anti-DNA Autoreactive B Lymphocytes by the Yaa Mutation
Authors: Moll Thomas , Martinez-Soria Eduardo , Santiago-Raber Marie-Laure , Amano Hirofumi , Pihlgren-Bosch Maria , Marinkovic Dragan , Izui Shozo
Published: April 2014

An as-yet-unidentified mutation, Y-linked autoimmune acceleration (Yaa), is responsible for the accelerated development of lupus-like autoimmune syndrome in mice. In view of a possible role for Yaa as a positive regulator of BCR signaling, we have explored whether the expression of the Yaa mutation affects the development and activation of transgenic autoreactive B cells expressing either 4C8 IgM anti-RBC or Sp6 IgM anti-DNA. In this study, we show that the expression of the Yaa mutation induced a lethal form of autoimmune hemolytic anemia in 4C8 transgenic C57BL/6 mice, likely as a result of activation of 4C8 anti-RBC autoreactive B cells early in life. This was further supported, although indirectly, by increased T cell-independent IgM production in spleens of nontransgenic C57BL/6 mice bearing the Yaa mutation. In contrast, Yaa failed to induce activation of Sp6 anti-DNA autoreactive B cells, consistent with a lack of increased IgM anti-DNA production in nontransgenic C57BL/6 Yaa mice. Our results suggest that Yaa can activate autoreactive B cells in a BCR-dependent manner, related to differences in the form and nature of autoantigens.
T Cells Undergo Rapid ON/OFF but Not ON/OFF/ON Cycling of Cytokine Production in Response to Antigen
Authors: Corbin Gail A. , Harty John T.
Published: April 2014

Inflammatory cytokines such as IFN-γ and TNF produced by Ag-stimulated CD4+ and CD8+ T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8+ T cells maintain IFN-γ production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8+ T cells could rapidly cycle IFN-γ production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8+ T cells, our results show that Listeria monocytogenes-specific CD4+ T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-γ and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4+ T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8+ T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8+ T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-γ after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-γ secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.
TLR Ligands Can Activate Dendritic Cells to Provide a MyD88-Dependent Negative Signal for Th2 Cell Development
Authors: Sun Jie , Walsh Matthew , Villarino Alejandro V. , Cervi Laura , Hunter Christopher A. , Choi Yongwon , Pearce Edward J.
Published: April 2014

During infection, CD4+ Th cell responses polarize to become primarily Th1 or Th2. Th1 cells, which make IFN-γ, are crucial for immunity to many bacterial and protozoal infections, whereas Th2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to helminth infections. Polarized Th1 responses are induced by dendritic cells (DCs), which respond to pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in Th1 cell outgrowth, and coordinately process and present Ag in the context of MHC class II to activate naive Th cells. In this study we show that in addition to providing positive signals for Th1 cell development, mouse DCs activated by TLR engagement can also provide a potent negative signal that prevents the development of Th2 cells. Production of this signal, which is not IL-12, IL-18, IL-23, IL-27, or IFN-γ and is not provided via Th1 cells, is dependent upon a MyD88-dependent, TNF receptor-associated factor-6-independent signaling pathway in DCs. The signal is released from DCs in response to activation via TLR ligands and exerts an effect directly on Th cells rather than through a third-party cell. Our findings indicate that DCs can provide potent negative as well as positive instruction for Th response polarization, and that these instructional signals are distinct and independent.
IFN-γ Negatively Modulates Self-Renewal of Repopulating Human Hemopoietic Stem Cells
Authors: Yang Liping , Dybedal Ingunn , Bryder David , Nilsson Lars , Sitnicka Ewa , Sasaki Yutaka , Jacobsen Sten Eirik W.
Published: April 2014

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-γ, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-γ might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-γ on HSC maintenance and function. Although purified cord blood CD34+CD38− cells underwent cell divisions in the presence of IFN-γ, cycling HSCs exposed to IFN-γ in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-γ accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-γ can negatively affect human HSC self-renewal.
Pharmacological Targeting of Anaphylatoxin Receptors during the Effector Phase of Allergic Asthma Suppresses Airway Hyperresponsiveness and Airway Inflammation
Authors: Baelder Ralf , Fuchs Barbara , Bautsch Wilfried , Zwirner Joerg , Köhl Jörg , Hoymann Heinz G , Glaab Thomas , Erpenbeck Veit , Krug Norbert , Braun Armin
Published: April 2014

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.
Role of CD28 in Polyclonal and Specific T and B Cell Responses Required for Protection against Blood Stage Malaria
Authors: Elias Rosa M. , Sardinha Luiz R. , Bastos Karina R. B. , Zago Cláudia A. , da Silva Ana Paula Freitas , Alvarez José M. , Lima Maria Regina D’Império
Published: April 2014

The role of B7/CD28 costimulatory pathway in the polyclonal and specific lymphocyte activation induced by blood stages of Plasmodium chabaudi AS was investigated in CD28 gene knockout (CD28−/−) and C57BL/6 (CD28+/+) mice. Analysis of the spleen during the acute infection revealed a similar increase in T and B cell populations in both groups of mice. Moreover, CD28−/− mice were able to develop a polyclonal IgM response to P. chabaudi. On the contrary, the polyclonal IgG2a response was markedly reduced in the absence of CD28. Production of IFN-γ; up-regulation of CD69, CD40L, CD95 (Fas), and CD95L (Fas ligand); and induction of apoptosis were also affected by the lack of CD28. Interestingly, the ability to control the first parasitemia peak was not compromised in acutely infected CD28−/− mice, but CD28−/− mice failed to eradicate the parasites that persisted in the blood for >3 mo after infection. In addition, drug-cured CD28−/− mice were unable to generate memory T cells, develop an anamnesic IgG response, or eliminate the parasites from a secondary challenge. The incapacity of CD28−/− mice to acquire a full protective immunity to P. chabaudi correlated with an impaired production of specific IgG2a. Moreover, reinfected CD28−/− mice were protected by the adoptive transfer of serum from reinfected CD28+/+ mice containing specific IgG2a. Our results demonstrate that the polyclonal lymphocyte response is only partially affected by the absence of CD28, but this coreceptor is essential to generate specific T and B cell responses required for complete protection against P. chabaudi malaria.
Proinflammatory Bacterial Peptidoglycan as a Cofactor for the Development of Central Nervous System Autoimmune Disease
Authors: Visser Lizette , Jan de Heer Hendrik , Boven Leonie A. , van Riel Debby , van Meurs Marjan , Melief Marie-José , Zähringer Ulrich , van Strijp Jos , Lambrecht Bart N. , Nieuwenhuis Edward E. , Laman Jon D.
Published: April 2014

Upon stimulation by microbial products through TLR, dendritic cells (DC) acquire the capacity to prime naive T cells and to initiate a proinflammatory immune response. Recently, we have shown that APC within the CNS of multiple sclerosis (MS) patients contain peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria, which signals through TLR and NOD. In this study, we report that Staphylococcus aureus PGN as a single component can support the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for MS. Mice immunized with an encephalitogenic myelin oligodendrocyte glycoprotein peptide in IFA did not develop EAE. In contrast, addition of PGN to the emulsion was sufficient for priming of autoreactive Th1 cells and development of EAE. In vitro studies demonstrate that PGN stimulates DC-mediated processes, reflected by increased Ag uptake, DC maturation, Th1 cell expansion, activation, and proinflammatory cytokine production. These data indicate that PGN-mediated interactions result in proinflammatory stimulation of Ag-specific effector functions, which are important in the development of EAE. These PGN-mediated processes may occur both within the peripheral lymph nodes as well as in the CNS and likely involve recognition by TLR on DC. Thus, PGN may provide a physiological trigger of DC maturation, and in this way disrupt the normal tolerance to self Ag. As such, PGN signaling pathways may serve as novel targets for the treatment of MS.