bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids Enhance or Spare Innate Immunity: Effects in Airway Epithelium Are Mediated by CCAAT/Enhancer Binding Proteins
Authors: Zhang Ning , Truong-Tran Quynh Ai , Tancowny Brian , Harris Kathleen E. , Schleimer Robert P.
Published: April 2014

Although it is widely accepted that glucocorticoids (GC) are a mainstay of the treatment of diseases characterized by airway inflammation, little is known about the effects of GC on local innate immunity. In this article, we report that respiratory epithelial cells manifested a local “acute phase response” after stimulation with TLR activation and TNF-α and that GC spared or enhanced the epithelial expression of molecules that are involved in host defense, including complement, collectins, and other antimicrobial proteins. As expected, GC inhibited the expression of molecules responsible for inflammation such as cytokines (IFNβ and GM-CSF) and chemokines (RANTES and IL-8). Studies using Western blotting, EMSA, and functional analysis indicated that the selective effects of GC are mediated through activation of the transcription factor C/EBP. Knockdown of C/EBPβ by small interfering RNA blocked the enhancement by GC of host defense molecule expression but had no effect on inflammatory gene expression. These results suggest that GC spare or enhance local innate host defense responses in addition to exerting anti-inflammatory actions. It is possible that the known ability of GC to reduce the exacerbation of diseases in which infectious organisms serve as triggering factors (e.g., asthma, allergic bronchopulmonary aspergillosis, and chronic obstructive pulmonary disease) may result in part from enhanced innate immune responses in airway mucosa.
Broad TCR Usage in Functional HIV-1-Specific CD8+ T Cell Expansions Driven by Vaccination during Highly Active Antiretroviral Therapy
Authors: Yang Hongbing , Dong Tao , Turnbull Emma , Ranasinghe Srinika , Ondondo Beatrice , Goonetilleke Nilu , Winstone Nicola , di Gleria Kati , Bowness Paul , Conlon Christopher , Borrow Persephone , Hanke Tomáš , McMichael Andrew , Dorrell Lucy
Published: April 2014

During chronic HIV-1 infection, continuing viral replication is associated with impaired proliferative capacity of virus-specific CD8+ T cells and with the expansion and persistence of oligoclonal T cell populations. TCR usage may significantly influence CD8+ T cell-mediated control of AIDS viruses; however, the potential to modulate the repertoire of functional virus-specific T cells by immunotherapy has not been explored. To investigate this, we analyzed the TCR Vβ usage of CD8+ T cells populations which were expanded following vaccination with modified vaccinia virus Ankara expressing a HIV-1 gag/multiepitope immunogen (MVA.HIVA) in HIV-1-infected patients receiving highly active antiretroviral therapy. Vaccinations induced the re-expansion of HIV-1-specific CD8+ T cells and these showed broad TCR Vβ usage which was maintained for at least 1 year in some individuals. By contrast, virus-specific CD8+ T cell populations in the same donors which failed to expand after vaccination and in unvaccinated controls were oligoclonal. Simultaneously, we observed that CD8+ T cells recognizing vaccine-derived HIV-1 epitopes displayed enhanced capacity to proliferate and to inhibit HIV-1 replication in vitro, following MVA.HIVA immunizations. Taken together, these data indicate that an attenuated viral-vectored vaccine can modulate adaptive CD8+ T cell responses to HIV-1 and improve their antiviral functional capacity. The potential therapeutic benefit of this vaccination approach warrants further investigation.
Chronic Sepsis Mortality Characterized by an Individualized Inflammatory Response
Authors: Osuchowski Marcin F. , Welch Kathy , Yang Huan , Siddiqui Javed , Remick Daniel G.
Published: April 2014

Late mortality in septic patients often exceeds the lethality occurring in acute sepsis, yet the immunoinflammatory alterations preceding chronic sepsis mortality are not well defined. We studied plasma cytokine concentrations preceding late septic deaths (days 6–28) in a murine model of sepsis induced by polymicrobial peritonitis. The late prelethal inflammatory response varied from a virtually nonexistent response in three of 14 to a mixed response in eight of 14 mice to the concurrent presence of nearly all measured cytokines, both proinflammatory and anti-inflammatory in three of 14 mice. In responding mice a consistent prelethal surge of plasma MIP-2 (1.6 vs 0.12 ng/ml in survivors; mean values), MCP-1 (2.0 vs 1.3 ng/ml), soluble TNF receptor type I (2.5 vs 0.66 ng/ml), and the IL-1 receptor antagonist (74.5 vs 3.3 ng/ml) was present, although there were infrequent increases in IL-6 (1.9 vs 0.03 ng/ml) and IL-10 (0.12 vs 0.04 ng/ml). For high mobility group box 1, late mortality was signaled by its decrease in plasma levels (591 vs 864 ng/ml). These results demonstrate that impeding mortality in the chronic phase of sepsis may be accurately predicted by plasma biomarkers, providing a mechanistic basis for individualized therapy. The pattern of late prelethal responses suggest that the systemic inflammatory response syndrome to compensatory anti-inflammatory response syndrome transition paradigm fails to follow a simple linear pattern.
Group V Phospholipase A2-Derived Lysophosphatidylcholine Mediates Cyclooxygenase-2 Induction in Lipopolysaccharide-Stimulated Macrophages
Authors: Ruipérez Violeta , Casas Javier , Balboa María A. , Balsinde Jesús
Published: April 2014

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A2 (sPLA2-V) was required for COX-2 induction, but the nature of the sPLA2-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA2-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA2-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA2-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA2-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.
Administration of Cyclooxygenase-2 Inhibitor Reduces Joint Inflammation but Exacerbates Osteopenia in IL-1α Transgenic Mice Due to GM-CSF Overproduction
Authors: Niki Yasuo , Takaishi Hironari , Takito Jiro , Miyamoto Takeshi , Kosaki Naoto , Matsumoto Hideo , Toyama Yoshiaki , Tada Norihiro
Published: April 2014

IL-1α transgenic (Tg) mice exhibit chronic inflammatory arthritis and subsequent osteopenia, with IL-1-induced GM-CSF playing an important role in the pathogenesis. This study analyzed the mechanisms underlying osteopenia in Tg mice, and the therapeutic effects of the cyclooxygenase-2 inhibitor celecoxib on such osteopenia. Inhibited osteoclast formation was observed in RANKL-treated bone marrow cell (BMC) cultures from Tg mice and coculture of Tg-derived BMCs and wild-type-derived primary osteoblasts (POBs). FACS analysis indicated that this inhibition was attributable to a decreased number of osteoclast precursors within Tg-derived BMCs. Moreover, in coculture of Tg-derived POBs and either Tg- or wild-type-derived BMCs, osteoclast formation was markedly inhibited because Tg-derived POBs produced abundant GM-CSF, known as a potent inhibitor of osteoclast differentiation. Histomorphometric analysis of Tg mice revealed that both bone formation and resorption were decreased, with bone formation decreased more prominently. Interestingly, administration of celecoxib resulted in further deterioration of osteopenia where bone formation was markedly suppressed, whereas bone resorption remained unchanged. These results were explained by our in vitro observation that celecoxib dose-dependently and dramatically decreased osteogenesis by Tg mouse-derived POBs in culture, whereas mRNA expressions of GM-CSF and M-CSF remained unchanged. Consequently, blockade of PGE2 may exert positive effects on excessively enhanced bone resorption observed in inflammatory bone disease, whereas negative effects may occur mainly through reduced bone formation, when bone resorption is constitutively down-regulated as seen in Tg mice.
Heterogeneity of Effector Phenotype for Acute Phase and Memory Influenza A Virus-Specific CTL
Authors: Jenkins Misty R. , Kedzierska Katherine , Doherty Peter C. , Turner Stephen J.
Published: April 2014

Ag-specific, CD8+ CTLs clear influenza A viruses from the lung via granzyme (Gzm) and perforin-dependent mechanisms. Ex vivo analysis of perforin-Gzm mRNA profiles demonstrated substantial heterogeneity in patterns of effector mRNA transcription of CD8+ DbNP366- or DbPA224-specific CTL. The only difference between the two epitope-specific sets was apparent very early after infection with similar molecular profiles seen in peak primary and secondary responses and in long-term memory. Surprisingly, memory T cells also expressed a diverse pattern of effector mRNA profile with an emphasis on GzmB and, surprisingly, GzmK. This analysis thus defines how naive, effector, and memory T cells differ in cytotoxic potential and provides novel insight into the molecular signatures of effector molecules observed at various stages after infection.
Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus
Authors: Ding Yong , Kantarci Alpdogan , Badwey John A. , Hasturk Hatice , Malabanan Alan , Van Dyke Thomas E.
Published: April 2014

The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-α and IL-1β) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.
Fullerene Nanomaterials Inhibit the Allergic Response
Authors: Ryan John J. , Bateman Henry R. , Stover Alex , Gomez Greg , Norton Sarah K. , Zhao Wei , Schwartz Lawrence B. , Lenk Robert , Kepley Christopher L.
Published: April 2014

Fullerenes are a class of novel carbon allotropes that may have practical applications in biotechnology and medicine. Human mast cells (MC) and peripheral blood basophils are critical cells involved in the initiation and propagation of several inflammatory conditions, mainly type I hypersensitivity. We report an unanticipated role of fullerenes as a negative regulator of allergic mediator release that suppresses Ag-driven type I hypersensitivity. Human MC and peripheral blood basophils exhibited a significant inhibition of IgE dependent mediator release when preincubated with C60 fullerenes. Protein microarray demonstrated that inhibition of mediator release involves profound reductions in the activation of signaling molecules involved in mediator release and oxidative stress. Follow-up studies demonstrated that the tyrosine phosphorylation of Syk was dramatically inhibited in Ag-challenged cells first incubated with fullerenes. In addition, fullerene preincubation significantly inhibited IgE-induced elevation in cytoplasmic reactive oxygen species levels. Furthermore, fullerenes prevented the in vivo release of histamine and drop in core body temperature in vivo using a MC-dependent model of anaphylaxis. These findings identify a new biological function for fullerenes and may represent a novel way to control MC-dependent diseases including asthma, inflammatory arthritis, heart disease, and multiple sclerosis.
Immune Complexes Inhibit Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells
Authors: Laborde Evangelina A. , Vanzulli Silvia , Beigier-Bompadre Macarena , Isturiz Martín A. , Ruggiero Raúl A. , Fourcade Mariano G. , Catalan Pellet Antonio C. , Sozzani Silvano , Vulcano Marisa
Published: April 2014

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcγRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcγR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcγRI and to a lesser extend with FcγRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.
Unc119 Regulates Myofibroblast Differentiation through the Activation of Fyn and the p38 MAPK Pathway
Authors: Vepachedu Ramarao , Gorska Magdalena M. , Singhania Neil , Cosgrove Gregory P. , Brown Kevin K. , Alam Rafeul
Published: April 2014

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of α-smooth muscle actin (α-SMA) and myofibroblast differentiation by TGF-β in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-β and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate α-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of α-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced α-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-β and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of α-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.
TGF-β1 Regulates Antigen-Specific CD4+ T Cell Responses in the Periphery
Authors: Robinson Richard T. , Gorham James D.
Published: April 2014

T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-β1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+ T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+ T cell expansion in TGF-β1−/− mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vβ repertoires in peripheral CD4+ T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+ T cells from 11-day-old TGF-β1−/− mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vβ CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-β1−/− CD4+ T cells than for TGF-β1+/− CD4+ T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-β1−/− CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-β1−/− mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-β1 in the peripheral regulation of Ag-specific CD4+ T cell responses.
The Inositol 5′-Phosphatase SHIP-2 Negatively Regulates IgE-Induced Mast Cell Degranulation and Cytokine Production
Authors: Leung Wai-Hang , Bolland Silvia
Published: April 2014

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells initiates signaling pathways leading to degranulation and cytokine release. It has been reported that SHIP-1 negatively regulates FcεRI-triggered pathways but it is unknown whether its homologous protein SHIP-2 has the same function. We have used a lentiviral-based RNA interference technique to obtain SHIP-2 knockdown bone marrow-derived mast cells (BMMCs) and have found that elimination of SHIP-2 results in both increased mast cell degranulation and cytokine (IL-4 and IL-13) gene expression upon FcεRI stimulation. Elimination of SHIP-2 from BMMCs has no effect on FcεRI-triggered calcium flux, tyrosine phosphorylation of MAPKs or in actin depolymerization following activation. Rather, we observe that absence of SHIP-2 results in increased activation of the small GTPase Rac-1 and in enhanced microtubule polymerization upon FcεRI engagement. Coimmunoprecipitation experiments in rat basophilic leukemia (RBL 2H3) cells show that SHIP-2 interacts with the FcεRI β-chain, Gab2 and Lyn and that unlike SHIP-1, it does not associate with SHC in mast cells. Our results report a negative regulatory role of SHIP-2 on mast cell activation that is calcium independent and distinct from the regulation by SHIP-1.
Tolerance Induction in Presensitized Bone Marrow Recipients by Veto CTLs: Effective Deletion of Host Anti-Donor Memory Effector Cells
Authors: Reich-Zeliger Shlomit , Bachar-Lustig Esther , Bar-Ilan Avichai , Reisner Yair
Published: April 2014

Veto cells have been defined as cells capable of inducing apoptosis of effector CD8 cells recognizing their disparate MHC Ags. Tolerance induced by donor-type veto cells is desirable, because it is restricted to depletion of anti-donor clones without depletion of other immune specificities. It has been shown that anti-third party CTLs exhibit marked veto activity with reduced capacity to induce graft-vs-host disease, when tested on naive effector cells. However, presensitized T cells could play an important role in graft rejection, and therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in bone marrow transplantation. To address this question, we compared naive and presensitized TCR transgenic effector CD8 T cells, bearing a TCR against H-2d. Both cell types exhibited similar predisposition to killing by veto CTLs in vitro, and this killing was dependent in both cell types on Fas-FasL signaling as shown by using Fas-deficient CD8 T cells from (lprx2c) F1 mice. When tested in a stringent mouse model, in which bone marrow rejection is mediated by adoptively transferred host type T cells into lethally irradiated recipients, veto CTLs were equally effective in overcoming rejection of naive or presensitized host T cells.
Temporal and Spatial Changes of Histone 3 K4 Dimethylation at the IFN-γ Gene during Th1 and Th2 Cell Differentiation
Authors: Hamalainen-Laanaya Heli K. , Kobie James J. , Chang Chawnshang , Zeng Wei-ping
Published: April 2014

Covalent modification of nucleosomal histones is an important mechanism for cytokine gene regulation in Th1 and Th2 cells. In this study, we analyzed the kinetics of histone H3 K4 dimethylation (H3K4me2) of the IFN-γ gene. Minimal levels of H3K4me2 were found in naive CD4 T cells. After 5 days of differentiation, H3K4me2 levels were elevated in both Th1 and Th2 cells at the −5.3 kb, the promoter, the intronic DNase I hypersensitive sites, and 3′ distal sites including the +9.5 kb and +16 kb sites. Th1 cells maintained high levels of H3K4me2 after longer time of culture. However, in Th2 cells after 14 days, high levels of H3K4me2 were detected only at the −5.3 kb and the promoter, whereas H3K4me2 was lost at the 3′ distal sites and greatly diminished at the DNase I hypersensitive sites. After 28 days, Th2 cells lose H3K4me2 at all sites. Unlike the long-term primary Th2 cells, the Th2 clone D10 showed strong H3K4me2 at the IFN-γ gene with distinctly high levels at the 3′ distal sites. CD4 T cells transgenic for Hlx or infected with T-bet-expressing retrovirus produced IFN-γ and retained high levels of H3K4me2 even after differentiated under Th2 polarizing conditions, suggesting positive roles of these two factors in maintaining high levels of H3K4me2 at the IFN-γ gene.
The Activating Immunoreceptor NKG2D and Its Ligands Are Involved in Allograft Transplant Rejection
Authors: Kim Jim , Chang Catherine K. , Hayden Tracy , Liu Feng-Chun , Benjamin Jonathan , Hamerman Jessica A. , Lanier Lewis L. , Kang Sang-Mo
Published: April 2014

Although the linkage between innate and adaptive immunity in transplantation has been recognized, the mechanisms underlying this cooperation remain to be fully elucidated. In this study, we show that early “danger” signals associated with transplantation lead to rapid up-regulation of NKG2D ligands. A second wave of NKG2D ligand up-regulation is mediated by the adaptive immune response to allografts. Treatment with an Ab to NKG2D was highly effective in preventing CD28-independent rejection of cardiac allografts. Notably, NKG2D blockade did not deplete CD8+ T cells or NK1.1+ cells nor affect their migration to the allografts. These results establish a functional role of NKG2D and its ligands in the rejection of solid organ transplants.
Preferential Migration of T Regulatory Cells Induced by IL-16
Authors: McFadden Caroline , Morgan Ross , Rahangdale Shilpa , Green Daniel , Yamasaki Hisato , Center David , Cruikshank William
Published: April 2014

As a natural ligand for CD4, IL-16 has been shown to preferentially induce migration in Th1 cells, and, in long-term cultures with IL-2, IL-16 facilitates the expansion of CD4+CD25+ cells. In addition, IL-16 has an immunomodulatory role in asthmatic inflammation, as exogenous administration significantly reduces inflammation and airway hyperreactivity. The mechanism for this, however, is not clear. Based on its functional characteristics and potential immunomodulatory role, we investigated the ability of IL-16 to recruit and influence the development of T regulatory (Treg) cells. We now demonstrate that IL-16 preferentially induces migration in a CD25+CTLA-4+ human T cell subset and that responding cells produce IFNγ and TGFβ but not IL-10. These cells are relatively unresponsive to antigenic stimulation and can suppress proliferation and IL-5, but not IFNγ, production by autologous T cells. We further demonstrate that IL-16-recruited cells are enriched for Forkhead box P3 (Foxp3). In addition, we find that IL-16 stimulation may facilitate de novo induction of Foxp3+ Treg cells, because the stimulation of FoxP3-negative T cells for 48 h results in the expression of FoxP3 mRNA and protein. These data indicate that at sites of inflammation IL-16 may contribute to selective Treg cell expansion through the preferential induction of a migratory response from existing Treg cells, as well as by the induction of de novo generation of FoxP3+ cells. These findings offer a potential mechanism for the immunosuppressive effects of IL-16 seen in Th2-mediated inflammation.
Leflunomide Inhibits PDK1/Akt Pathway and Induces Apoptosis of Human Mast Cells
Authors: Sawamukai Norifumi , Saito Kazuyoshi , Yamaoka Kunihiro , Nakayamada Shingo , Ra Chisei , Tanaka Yoshiya
Published: April 2014

Mast cells release many inflammatory mediators that play an important role not only in allergic diseases but also in chronic inflammatory diseases, autoimmune diseases, and others. A lot of mast cells exist in synovium of rheumatoid arthritis, and it is known that synovitis does not occur in mast cell-deficient mice. Thus, it is thought that mast cells play a very important role in rheumatoid arthritis pathogenesis. Leflunomide is a drug used clinically in the treatment of rheumatoid arthritis. We used clinical doses of 2-cyano-3-hydroxy-N-(4-trifluoromethylphenyl)-butenamide (A77 1726), which is an active metabolite of leflunomide, and decreased the number of viable human primary mast cells in a concentration-dependent manner. This decrease was not reversed by uridine. Inhibition of pyrimidine synthesis by dihydro-orotic acid dehydrogenase inhibition, which is the primary mechanism of action of A77 1726, was not involved. A77 1726 dramatically induced apoptosis of human mast cells and inhibited the phosphorylation of Akt, an important survival signal of mast cells, in a concentration-dependent manner. Caspases 3 and 9, downstream molecules of Akt survival pathway, were also fragmented by A77 1726. In addition, it became evident for the first time that the mechanism involved in this result was the concentration-dependent inhibition of PDK1 phosphorylation, which controls the activation of Akt. These results indicate a new way of controlling mast cells and may therefore be the basis for innovative approaches to the treatment of various diseases related to mast cells.
Requirement for Both JAK-Mediated PI3K Signaling and ACT1/TRAF6/TAK1-Dependent NF-κB Activation by IL-17A in Enhancing Cytokine Expression in Human Airway Epithelial Cells
Authors: Huang Fei , Kao Cheng-Yuan , Wachi Shinichiro , Thai Philip , Ryu Jisu , Wu Reen
Published: April 2014

Through DNA microarray analysis and quantitative PCR verification, we have identified additional IL-17A-inducible genes—IL-19, CXCL-1, -2, -3, -5, and -6—in well-differentiated normal human bronchial epithelial cells. These genes, similar to previously described human β-defensin-2 (HBD-2) and CCL-20, were induced by a basolateral treatment of IL-17A, and regulated by PI3K signaling and NF-κB activation. For PI3K signaling, increases of cellular PIP3 and phosphorylation of downstream molecules, such as Akt and glycogen synthase kinase-3β (GSK3β) (S9), were detected. Induced gene expression and HBD-2 promoter activity were attenuated by LY294002, p110α small-interfering RNA (siRNA), as well as by an overexpression of constitutively active GSK3β(S9A) or wild-type phosphatase and tensin homolog. Increased phosphorylation of JAK1/2 after IL-17A treatment was detected in primary normal human bronchial epithelium cells. Transfected siRNAs of JAK molecules and JAK inhibitor I decreased IL-17A-induced gene expression and GSK3β(S9) phosphorylation. However, both JAK inhibitor I and PI3K inhibitor had no effect on the DNA-binding activities of p65 and p50 to NF-κB consensus sequences. This result suggested a JAK-associated PI3K signaling axis is independent from NF-κB activation. With siRNA to knockdown STIR (similar expression to fibroblast growth factor and IL-17R; Toll-IL-1R)-related signaling molecules, such as Act1, TNFR-associated factor 6 (TRAF6), and TGF-β-activated kinase 1 (TAK1), and transfection of A52R, an inhibitor of the MyD88/TRAF6 complex, or dominant-negative TAK1, IL-17A-inducible gene expression and HBD-2 promoter activity were reduced. Additionally, IL-17A-induced p65 and p50 NF-κB activations were confirmed and their nuclear translocations were down-regulated by siRNAs of TRAF6 and TAK1. These results suggest that two independent and indispensable signaling pathways—1) JAK1-associated PI3K signaling and 2) Act1/TRAF6/TAK1-mediated NF-κB activation—are stimulated by IL-17A to regulate gene induction in human airway epithelial cells.
Naive and Memory T Cells Induce Different Types of Graft-versus-Host Disease
Authors: Dutt Suparna , Tseng Diane , Ermann Joerg , George Tracy I. , Liu Yin Ping , Davis Corrine R. , Fathman C. Garrison , Strober Samuel
Published: April 2014

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4+ naive (CD62LhighCD44low) T cells and CD4+ effector memory (CD62LlowCD44high) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-γ in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.
Spontaneous Class Switch Recombination in B Cell Lymphopoiesis Generates Aberrant Switch Junctions and Is Increased after VDJ Rearrangement
Authors: Edry Efrat , Koralov Sergei B. , Rajewsky Klaus , Melamed Doron
Published: April 2014

Mature B cells replace the μ constant region of the H chain with a downstream isotype in a process of class switch recombination (CSR). Studies suggest that CSR induction is limited to activated mature B cells in the periphery. Recently, we have shown that CSR spontaneously occur in B lymphopoiesis. However, the mechanism and regulation of it have not been defined. In this study, we show that spontaneous CSR occurs at all stages of B cell development and generates aberrant joining of the switch junctions as revealed by: 1) increased load of somatic mutations around the CSR break points, 2) reduced sequence overlaps at the junctions, and 3) excessive switch region deletion. In addition, we found that incidence of spontaneous CSR is increased in cells carrying VDJ rearrangements. Our results reveal major differences between spontaneous CSR in developing B cells and CSR induced in mature B cells upon activation. These differences can be explained by deregulated expression or function of activation-induced cytidine deaminase early in B cell development.
Donor Bone Marrow Type II (Non-Vα14Jα18 CD1d-Restricted) NKT Cells Suppress Graft-Versus-Host Disease by Producing IFN-γ and IL-4
Authors: Kim Ji Hyung , Choi Eun Young , Chung Doo Hyun
Published: April 2014

NKT cells in donor bone marrow (BM) have been demonstrated to protect against graft-vs-host disease (GVHD) following BM transplantation. Murine NKT cells are divided into two distinct subsets based on the invariant Vα14Jα18 TCR expression. However, details of the subset and mechanisms of the BM NKT cells involved in suppressing GVHD have not been clarified. Irradiated BALB/c or C3H/HeN mice administered B6 or Jα18−/− BM cells show attenuation of GVHD, whereas recipients given CD1d−/− BM cells did not show attenuation. Moreover, coinjection of BM non-Vα14Jα18 CD1d-restricted (type II) NKT cells and CD1d−/− BM cells suppressed GVHD, whereas coinjection of BM Vα14Jα18 TCR (type I) NKT cells did not. These protective effects on GVHD depended upon IFN-γ-producing type II NKT cells, which induced the apoptosis of donor T cells. The splenocytes of mice administered BM cells from B6.IL-4−/− or Jα18−/−IL-4−/− mice produced lower levels of IL-4 and IL-10 than the splenocytes of mice transplanted with BM cells from B6, B6.IFN-γ−/−, Jα18−/−, or Jα18−/−IFN-γ−/− mice. Taken together, our results show that IFN-γ-producing BM type II NKT cells suppress GVHD by inducing the apoptosis of donor T cells, while IL-4-producing BM type II NKT cells protect against GVHD by deviating the immune system toward a Th2-type response.
CD8 T Cell Help for Innate Antitumor Immunity
Authors: Shanker Anil , Verdeil Grégory , Buferne Michel , Inderberg-Suso Else-Marit , Puthier Denis , Joly Florence , Nguyen Catherine , Leserman Lee , Auphan-Anezin Nathalie , Schmitt-Verhulst Anne-Marie
Published: April 2014

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential “help” to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.
Primary Development and Participation in a Foreign Antigen-Driven Immune Response of a Chromatin-Reactive B Cell Clonotype Are Not Influenced by TLR9 or Other MyD88-Dependent TLRs
Authors: Coffey Francis , Liu Xiaohe , Manser Tim
Published: April 2014

Recent findings support a central role for TLRs in both foreign Ag-driven immune responses and systemic autoimmune diseases mediated by B lymphocytes. In vitro studies have shown that the Ag receptors (BCRs) on B cells specific for nuclear autoantigens can facilitate the delivery of these autoantigens to the endocytic compartment, resulting in activation of the nucleic acid-specific TLRs present in this subcellular locale. If this pathway is operative in vivo it might promote the development, survival, or activation of such autoreactive B cells. To test this idea, we evaluated the influence of a deficiency in the CpG DNA-specific TLR, TLR9, or all MyD88-dependent TLRs on the primary development and foreign Ag-driven immune response of B cells in a line of VH knockin mice that contains a high frequency of “dual reactive” B cells specific for DNA-based autoantigens such as chromatin, as well as the hapten arsonate. We found that although development and activation of these B cells in vitro are clearly influenced by DNA-based autoantigens, TLR9 or MyD88 deficiencies had no apparent effect on the primary development and participation in the anti-arsonate response of these B cells in vivo. We discuss these results in the context of previous models for the role of TLR9 and other TLRs in the regulation of antinuclear Ag B cell development and activity.
Dual Roles of IL-15 in Maintaining IL-7RαlowCCR7− Memory CD8+ T Cells in Humans via Recovering the Phosphatidylinositol 3-Kinase/AKT Pathway
Authors: Kim Hang-Rae , Hwang Kyung-A , Kang Insoo
Published: April 2014

Recently, we identified two subsets of CCR7− memory CD8+ T cells expressing high and low levels of the IL-7R α-chain (IL-7Rα) that is essential for memory T cell survival in human peripheral blood. IL-7RαlowCCR7− memory CD8+ T cells that produce effector cytokines and perforin have impaired proliferation and survival in response to TCR triggering and IL-7, respectively. These findings raise a question of how such cells are sustained at significant numbers, >20% of peripheral CD8+ T cells, despite impaired IL-7- and TCR-mediated cell maintenance. In this study, we demonstrate that IL-7RαlowCCR7− memory CD8+ T cells have increased expression of IL-2/15R β-chain (IL-2/15Rβ), which is critical for IL-15 signaling, with enhanced gene expression of T box expressed in T cells (T-bet) and eomesodermin (eomes), transcriptional factors involved in IL-2/15Rβ expression compared with IL-7RαhighCCR7− memory CD8+ T cells. Such a cytokine chain is functional as IL-7RαlowCCR7− memory CD8+ T cells proliferate considerably in response to IL-15. Furthermore, adding IL-15 to TCR triggering recovers impaired TCR-mediated proliferation of IL-7Rαlow memory CD8+ T cells via restoring the activation of the PI3K/AKT pathway. These findings indicate that IL-15 has dual roles in maintaining IL-7RαlowCCR7− memory CD8+ T cells via TCR-dependent and -independent mechanisms. Moreover, IL-15 can be useful in reviving impaired proliferative function of such memory CD8+ T cells with effector functions against infections and tumors via rescuing the PI3K/AKT pathway.
Shaping of the Autoreactive Regulatory T Cell Repertoire by Thymic Cortical Positive Selection
Authors: Ribot Julie , Enault Geneviève , Pilipenko Sylvie , Huchenq Anne , Calise Maryline , Hudrisier Denis , Romagnoli Paola , van Meerwijk Joost P. M.
Published: April 2014

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4+CD25+Foxp3+ regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.
Sulfated Glycosphingolipid as Mediator of Phagocytosis: SM4s Enhances Apoptotic Cell Clearance and Modulates Macrophage Activity
Authors: Popovic Zoran V. , Sandhoff Roger , Sijmonsma Tjeerd P. , Kaden Sylvia , Jennemann Richard , Kiss Eva , Tone Edgar , Autschbach Frank , Platt Nick , Malle Ernst , Gröne Hermann-Josef
Published: April 2014

Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-β1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.
Localization and Differential Expression of Activation-Induced Cytidine Deaminase in the Amphibian Xenopus upon Antigen Stimulation and during Early Development
Authors: Marr Shauna , Morales Heidi , Bottaro Andrea , Cooper Michelle , Flajnik Martin , Robert Jacques
Published: April 2014

As in mammals, B cell maturation in the amphibian Xenopus involves somatic hypermutation (SHM) and class switch recombination to diversify the B cell receptor repertoire in response to Ag stimulation. Unlike mammals, however, the resulting increase in Ab affinity is poor in Xenopus, which is possibly related to the absence of germinal centers and a suboptimal selection mechanism of SHM. In mammals, both SHM and class switch recombination are mediated by the activation-induced cytidine deaminase enzyme and under Ag-dependent regulation. Given its evolutionary conservation in jawed vertebrates, we used activation-induced cytidine deaminase as a marker to monitor and localize B cell maturation in Xenopus upon immune responses and during early development. In adult, Xenopus laevis AID (XlAID) was detected mainly in the spleen, where cells expressing XlAID were preferentially distributed in follicular B cell zones, although some XlAID+ cells were also found in the red pulp. XlAID was markedly up-regulated in the spleen with different kinetics upon bacterial stimulation and viral infection. However, during secondary anti-viral response XlAID was also noticeably expressed by PBLs, suggesting that XlAID remains active in a subset of circulating B cells. During ontogeny, XlAID expression was detected as early as 5 days postfertilization in liver before the first fully differentiated B cells appear. Concomitant with appearance of mature B cells XlAID was up-regulated upon bacterial stimulation or viral infection at later larval stages. This study highlights the conserved involvement of XlAID during Ag-dependent B cell responses in Xenopus but also suggests another role in B cell differentiation earlier in ontogeny.
IL-6 Contributes to the Expression of RAGs in Human Mature B Cells
Authors: Hillion Sophie , Dueymes Maryvonne , Youinou Pierre , Jamin Christophe
Published: April 2014

Mature B cells acquire the capacity to revise rearranged Ig V region genes in secondary lymphoid organs. In previous studies, we demonstrated that cross-linking the BCR and the CD40 induces the expression of the RAG1 and RAG2 enzymes and, thereby, secondary rearrangements. We examine herein the mechanism that underpins RAG1 and RAG2 expression in peripheral and tonsil B cells. Coordinated engagement of the BCR and CD40 promoted the synthesis of IL-6 and, thereby, up-regulation of its receptor on activated B lymphocytes. Furthermore, we provide evidence that IL-6 initiates the expression of RAGs in circulating B cells, and extends those in tonsil B cells. Thus, neutralization of IL-6 or blocking of its receptor inhibits RAG expression. Moreover, we demonstrate that IL-6 impedes BCR-mediated termination of RAG gene expression in both population of B cells. The recovered inhibition of RAG gene transcription by IL-6 receptor blockade supports the notion that once recombination is launched, its termination is also regulated by IL-6. Taken together, these studies provide new insight into the dual role of IL-6 in inducing and terminating expression of the recombinase machinery for secondary rearrangements in mature human B cells.
Developmental Stage-Dependent Collaboration between the TNF Receptor-Associated Factor 6 and Lymphotoxin Pathways for B Cell Follicle Organization in Secondary Lymphoid Organs
Authors: Qin Junwen , Konno Hiroyasu , Ohshima Daisuke , Yanai Hiromi , Motegi Hidehiko , Shimo Yusuke , Hirota Fumiko , Matsumoto Mitsuru , Takaki Satoshi , Inoue Jun-ichiro , Akiyama Taishin
Published: April 2014

Signal transduction pathways regulating NF-κB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-κB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) β receptor (LtβR) signal induction of nonclassical NF-κB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtβR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.