bluuebunny/crossref_metadata_2025 huggingface dataset:
Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-κB Activation and Inducible NO Synthase Expression
Authors: Torgler Ralph , Bongfen Silayuv E. , Romero Jackeline C. , Tardivel Aubry , Thome Margot , Corradin Giampietro
Published: April 2014

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. Furthermore, no NF-κB activation was observed when Spect−/− parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88−/− mice showed no NF-κB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection.
Lack of Toll IL-1R8 Exacerbates Th17 Cell Responses in Fungal Infection
Authors: Bozza Silvia , Zelante Teresa , Moretti Silvia , Bonifazi Pierluigi , DeLuca Antonella , D’Angelo Carmen , Giovannini Gloria , Garlanda Cecilia , Boon Louis , Bistoni Francesco , Puccetti Paolo , Mantovani Alberto , Romani Luigina
Published: April 2014

TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8−/− mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8−/− mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-β in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI−/− mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.
SOCS-1 Protects against Chlamydia pneumoniae-Induced Lethal Inflammation but Hampers Effective Bacterial Clearance
Authors: Yang Tangbin , Stark Patrik , Janik Katrin , Wigzell Hans , Rottenberg Martin E.
Published: April 2014

Suppressor of cytokine signaling 1 (SOCS1) plays a major role in the inhibition of STAT1-mediated responses. STAT1-dependent responses are critical for resistance against infection with Chlamydia pneumoniae. We studied the regulation of expression of SOCS1 and SOCS3, and the role of SOCS1 during infection with C. pneumoniae in mice. Bone marrow-derived macrophages (BMM) and dendritic cells in vitro or lungs in vivo all showed enhanced STAT1-dependent SOCS1 mRNA accumulation after infection with C. pneumoniae. Infection-increased SOCS1 mRNA levels were dependent on IFN-αβ but not on IFN-γ. T or B cells were not required for SOCS1 mRNA accumulation in vivo. Infection-induced STAT1-phosphorylation occurred more rapidly in SOCS1−/− BMM. In agreement, expression of IFN-γ responsive genes, but not IL-1β, IL-6, or TNF-α were relatively increased in C. pneumoniae-infected SOCS1−/− BMM. Surprisingly, C. pneumoniae infection-induced IFN-α, IFN-β, and IFN-γ expression in BMM were attenuated by SOCS1. C. pneumoniae infection of RAG1−/−/SOCS1−/− mice induced a rapid lethal inflammation, accompanied by diminished pulmonary bacterial load and increased levels of iNOS and IDO but not IL-1β, IL-6, or TNF-α mRNA. In summary, C. pneumoniae infection induces a STAT1, IFN-αβ-dependent and IFN-γ independent SOCS1 mRNA accumulation. Presence of SOCS1 controls the infection-induced lethal inflammatory disease but impairs the bacterial control.
Functional CD8+ T Cell Responses in Lethal Ebola Virus Infection
Authors: Bradfute Steven B. , Warfield Kelly L. , Bavari Sina
Published: April 2014

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4+ and CD8+ T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8+ T cell IFN-γ responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8+ T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8+ cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the ‘impaired’ immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.
Deoxynucleic Acids from Cryptococcus neoformans Activate Myeloid Dendritic Cells via a TLR9-Dependent Pathway
Authors: Nakamura Kiwamu , Miyazato Akiko , Xiao Gang , Hatta Masumitsu , Inden Ken , Aoyagi Tetsuji , Shiratori Kohei , Takeda Kiyoshi , Akira Shizuo , Saijo Shinobu , Iwakura Yoichiro , Adachi Yoshiyuki , Ohno Naohito , Suzuki Kazuo , Fujita Jiro , Kaku Mitsuo , Kawakami Kazuyoshi
Published: April 2014

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9−/−) mice and MyD88−/− mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9−/− BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9−/− mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-κB in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.
Functional Adaptation of Nef to the Immune Milieu of HIV-1 Infection In Vivo
Authors: Lewis Martha J. , Balamurugan Arumugam , Ohno Ayako , Kilpatrick Stephanie , Ng Hwee L. , Yang Otto O.
Published: April 2014

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4+ T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.
Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery
Authors: Kushwah Rahul , Cao Huibi , Hu Jim
Published: April 2014

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with ∼6% of naive CD4+ T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4+ and CD8+ T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8+ T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.
Deviation from a Strong Th1-Dominated to a Modest Th17-Dominated CD4 T Cell Response in the Absence of IL-12p40 and Type I IFNs Sustains Protective CD8 T Cells
Authors: Orgun Nural N. , Mathis Meredith A. , Wilson Christopher B. , Way Sing Sing
Published: April 2014

The differentiation of naive CD4 T cells into specific effector subsets is controlled in large part by the milieu of cytokines present during their initial encounter with Ag. Cytokines that drive differentiation of the newly described Th17 lineage have been characterized in vitro, but the cytokines that prime commitment to this lineage in response to infection in vivo are less clear. Listeria monocytogenes (Lm) induces a strong Th1 response in wild-type mice. By contrast, we demonstrate that in the absence of IL-12p40 (or IFN-γ) and type I IFN receptor signaling, the Th1 Ag-specific CD4 T cell response is virtually abolished and replaced by a relatively low magnitude Th17-dominated response. This Th17 response was dependent on TGF-β and IL-6. Despite this change in CD4 T cell response, neither the kinetics of the CD4 and CD8 T cell responses, the quality of the CD8 T cell response, nor the ability of CD8 T cells to mediate protection were affected. Thus, generation of protective CD8 T cell immunity was resilient to perturbations that replace a strong Th1-dominated to a reduced magnitude Th17-dominated Ag-specific CD4 T cell response.
IFN-β Increases Listeriolysin O-Induced Membrane Permeabilization and Death of Macrophages
Authors: Zwaferink Heather , Stockinger Silvia , Hazemi Parastoo , Lemmens-Gruber Rosa , Decker Thomas
Published: April 2014

Type I IFN (IFN-I) signaling is detrimental to cells and mice infected with Listeria monocytogenes. In this study, we investigate the impact of IFN-I on the activity of listeriolysin O (LLO), a pore-forming toxin and virulence protein released by L. monocytogenes. Treatment of macrophages with IFN-β increased the ability of sublytic LLO concentrations to cause transient permeability of the plasma membrane. At higher LLO concentrations, IFN-β enhanced the complete breakdown of membrane integrity and cell death. This activity of IFN-β required Stat1. Perturbation of the plasma membrane by LLO resulted in activation of the p38MAPK pathway. IFN-β pretreatment enhanced LLO-mediated signaling through this pathway, consistent with its ability to increase membrane damage. p38MAPK activation in response to LLO was independent of TLR4, a putative LLO receptor, and inhibition of p38MAPK neither enhanced nor prevented LLO-induced death. IFN-β caused cells to express increased amounts of caspase 1 and to produce a detectable caspase 1 cleavage product after LLO treatment. Contrasting recent reports with another pore-forming toxin, this pathway did not aid cell survival as caspase1-deficient cells were equally sensitive to lysis by LLO. Key lipogenesis enzymes were suppressed in IFN-β-treated cells, which may exacerbate the membrane damage caused by LLO.
Mannose-Binding Lectin (MBL) Facilitates Opsonophagocytosis of Yeasts but Not of Bacteria despite MBL Binding
Authors: Brouwer Nannette , Dolman Koert M. , van Houdt Michel , Sta Marleen , Roos Dirk , Kuijpers Taco W.
Published: April 2014

Mannose-binding lectin (MBL) is a serum protein of the innate immune system. After binding to a microorganism, MBL in complex with MBL-associated serine proteases activates the complement system, resulting in cleavage of complement factor C3. Cleaved C3 on the surface of the microorganism mediates opsonization for clearance, but the impact of MBL on subsequent phagocytosis has not been widely studied. We investigated the role of MBL in complement activation and phagocytosis of various bacteria and yeast species by flow cytometry. We measured both the C3 deposition during serum opsonization of fluorescent-labeled microorganisms as well as subsequent uptake of these microorganisms by human neutrophils. In MBL-deficient sera, a consistently decreased C3 deposition on both zymosan and Candida albicans was found and a reduced phagocytosis by neutrophils that was restored by exogenous MBL. This indicates that the lectin pathway of complement activation is important for the opsonophagocytosis of yeasts. In contrast, the C1q-dependent classical pathway dominated in the opsonization and phagocytosis of Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, whereas no effect of MBL was found. Both the lectin and the classical pathway of complement activation were highly amplified by the alternative route for opsonophagocytosis by neutrophils of yeast as well as microbial species. In summary, our data demonstrate that yeast species are preferentially opsonized and subsequently phagocytosed via activation of the lectin pathway of complement, whereas the uptake of bacterial strains was found to be largely MBL independent.
Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways
Authors: Xu Lixin , Fang Hui , Frucht David M.
Published: April 2014

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1–4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.
Endogenous IFN-γ Production Is Induced and Required for Protective Immunity against Pulmonary Chlamydial Infection in Neonatal Mice
Authors: Jupelli Madhulika , Guentzel M. Neal , Meier Patricia A. , Zhong Guangming , Murthy Ashlesh K. , Arulanandam Bernard P.
Published: April 2014

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12–30) compared with BALB/c pups (days 9–12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-γ, not IL-4, production. Additionally, elevated IFN-γ, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-γ or IFN-γ receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-γ production, and that endogenous IFN-γ is important in protection against this infection. The enhanced IFN-γ induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.
ERK5/BMK1 Is Indispensable for Optimal Colony-Stimulating Factor 1 (CSF-1)-Induced Proliferation in Macrophages in a Src-Dependent Fashion
Authors: Rovida Elisabetta , Spinelli Elena , Sdelci Sara , Barbetti Valentina , Morandi Andrea , Giuntoli Serena , Dello Sbarba Persio
Published: April 2014

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-γ and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.
Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation
Authors: Pazdrak Konrad , Young Travis W. , Stafford Susan , Olszewska-Pazdrak Barbara , Straub Christof , Starosta Vitaliy , Brasier Allan , Kurosky Alexander
Published: April 2014

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor β-chain (GMRβ) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480–488) showed binding to Shp2 phosphatase and GMRβ. However, the interaction of GMRβ with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRβ with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRβ. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-α. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.
Dual Modulation of Airway Smooth Muscle Contraction by Th2 Cytokines via Matrix Metalloproteinase-1 Production
Authors: Ohta Yoshinori , Hayashi Masayuki , Kanemaru Takaaki , Abe Kihachiro , Ito Yushi , Oike Masahiro
Published: April 2014

Altered contractility of airway smooth muscle (SM) is one of the main causes of allergic asthma, in which the predominance of Th2 over Th1 cytokines plays a central role. In the present study, we examine the effects of Th2 cytokines on airway SM contraction. Treatment with a low concentration of IL-4 (0.2 ng/ml) for 6 h augmented, whereas higher concentrations (2–20 ng/ml) inhibited, agonist-induced contractions of collagen gels containing bovine tracheal SM cells. Another Th2 cytokine (IL-13) showed an augmentation of gel contraction in the concentration range of 20–200 ng/ml. IL-4 and IL-13 increased mRNA expression and protein secretion of matrix metalloproteinase (MMP)-1, but these cytokines did not affect Ca2+-mobilizing properties and phosphorylation levels of myosin L chain in bovine tracheal SM cells. These changes were sensitive to wortmannin, an inhibitor of PI3K, but not to leflunomide, an inhibitor of STAT6. Scanning electron microscope observation revealed that collagen fibers twining around SM cells were completely dissolved in 20 ng/ml IL-4-treated gels and reorganized into basket-like structure in 20 ng/ml IL-13-treated gels. Exogenous application of high and low concentrations of MMP-1 also induced the inhibition and augmentation of gel contraction, respectively. Furthermore, nonselective MMP inhibitor galardin suppressed the effects of IL-4 and IL-13 on gel contraction, and MMP-1-targeted small-interfering RNA reversed the inhibitory effects of IL-4 on gel contraction to the augmentation. This indicates that Th2 cytokines modulate airway contraction without affecting cellular contractility but by secreting MMP-1 from the SM cells via PI3K activation and changing cell-to-matrix interactions.
STAT-1 Signaling in Human Lung Fibroblasts Is Induced by Vanadium Pentoxide through an IFN-β Autocrine Loop
Authors: Antao-Menezes Aurita , Turpin Elizabeth A. , Bost Phillip C. , Ryman-Rasmussen Jessica P. , Bonner James C.
Published: April 2014

The inhalation of vanadium pentoxide (V2O5) results in bronchitis and airway fibrosis. The lung fibrotic response to V2O5 partially resolves where fibroblasts first proliferate and deposit collagen, but then undergo growth arrest and apoptosis. STAT-1 mediates fibroblast growth arrest and apoptosis. We previously reported that STAT-1 is a protective factor and mice lacking STAT-1 are more susceptible to lung fibrosis. We also reported that V2O5-induced STAT-1 phosphorylation in lung fibroblasts requires H2O2 and de novo protein synthesis. In this study, we identified IFN-β as the protein that mediates STAT-1 activation by V2O5 in normal human lung fibroblasts and identified NADPH and xanthine oxidase systems as sources of H2O2 that drive IFN-β gene expression. STAT-1 phosphorylation was decreased with neutralizing Abs to IFN-β as well as an inhibitor of JAK. V2O5 also increased transcription of an IFN-inducible and STAT-1-dependent chemokine, CXCL10. Inhibition of H2O2-generating enzyme systems NADPH oxidase by apocynin and xanthine oxidase by allopurinol individually reduced STAT-1 phosphorylation. Apocynin and allopurinol also decreased V2O5-induced IFN-β mRNA levels and CXCL10 expression. IFN-α transcription was inhibited only by allopurinol. Taken together, these data indicate that fibroblasts play a role in the innate immune response to vanadium-induced oxidative stress by synthesizing IFN-β and activating STAT-1 to cause growth arrest and increase levels of CXCL10, a potent antifibrotic factor. This mechanism is postulated to counterbalance profibrogenic mechanisms that follow V2O5 injury.
Genetic Analysis of the Role of the PI3K-Akt Pathway in Lipopolysaccharide-Induced Cytokine and Tissue Factor Gene Expression in Monocytes/Macrophages
Authors: Luyendyk James P. , Schabbauer Gernot A. , Tencati Michael , Holscher Todd , Pawlinski Rafal , Mackman Nigel
Published: April 2014

LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-κB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85α regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTENflox/flox/LysMCre mice (PTEN−/−), which have increased Akt activity. Analysis of LPS signaling in Pik3r1−/− and PTEN−/− cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IκBα or NF-κB nuclear translocation. LPS induction of TNF-α, IL-6, and tissue factor gene expression was increased in Pik3r1−/− peritoneal exudate cells and decreased in PTEN−/− peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1−/− bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85α subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.
A Helminth Immunomodulator Reduces Allergic and Inflammatory Responses by Induction of IL-10-Producing Macrophages
Authors: Schnoeller Corinna , Rausch Sebastian , Pillai Smitha , Avagyan Angela , Wittig Bianca M. , Loddenkemper Christoph , Hamann Alf , Hamelmann Eckard , Lucius Richard , Hartmann Susanne
Published: April 2014

The coincidence between infections with parasitic worms and the reduced prevalence of allergic disease in humans and in animal models has prompted the search for helminth molecules with antiallergic and antiinflammatory potential. We report herein that filarial cystatin, a secreted protease inhibitor of filarial nematodes, suppresses Th2-related inflammation and the ensuing asthmatic disease in a murine model of OVA-induced allergic airway responsiveness. Treatment with recombinant filarial cystatin inhibited eosinophil recruitment, reduced levels of OVA-specific and total IgE, down-regulated IL-4 production, and suppressed allergic airway hyperreactivity when applied during or after sensitization and before challenge with the allergen. Depletion of macrophages by clodronate-containing liposomes prevented the curative effects and restored the levels of infiltrating cells, IgE, and allergic airway reactivity. Blocking of IL-10 by application of anti-IL-10 receptor Abs restored the reduced number of infiltrating cells and the levels of OVA-specific IgE. In contrast, depletion of regulatory T cells by anti-CD25 Abs had only limited effects. Cystatin also modulated macrophage-mediated inflammation in a murine model of dextran sulfate sodium-induced colitis, leading to reduction of inflammatory infiltrations and epithelial damage. Our data demonstrate that treatment with a single helminth protein can exert the antiallergic effects of helminth infections.
A Critical Role for ABCG1 in Macrophage Inflammation and Lung Homeostasis
Authors: Wojcik Allison J. , Skaflen Marcus D. , Srinivasan Suseela , Hedrick Catherine C.
Published: April 2014

ATP-binding cassette transporter G1 (ABCG1) effluxes cholesterol from macrophages and plays an important role in pulmonary lipid homeostasis. We hypothesize that macrophages from Abcg1−/− mice have increased inflammatory activity, thereby promoting acceleration of pulmonary disease. We herein demonstrate increased numbers of inflammatory cytokines and infiltrating neutrophils, eosinophils, dendritic cells, T cells, and B cells into lungs of Abcg1−/− mice before the onset of severe lipidosis. We further investigated the role of macrophages in causing pulmonary disease by performing bone marrow transplantations using B6 and Abcg1−/− bone marrow. We found that it was the macrophage, and not pneumocyte type II cells or other nonhematopoietic cells in the lung, that appeared to be the primary cell type involved in the onset of both pulmonary lipidosis and inflammation in the Abcg1−/− mice. Additionally, our results demonstrate that Abcg1−/− macrophages had elevated proinflammatory cytokine production, increased apoptotic cell clearance, and were themselves more prone to apoptosis and necrosis. However, they were quickly repopulated by monocytes that were recruited to Abcg1−/− lungs. In conclusion, we have shown that ABCG1 deletion in macrophages causes a striking inflammatory phenotype and initiates onset of pulmonary lipidosis in mice. Thus, our studies reveal a critical role for macrophage ABCG1 in lung inflammation and homeostasis.
Delivery of Biologically Active Anti-Inflammatory Cytokines IL-10 and IL-1ra In Vivo by the Shigella Type III Secretion Apparatus
Authors: Chamekh Mustapha , Phalipon Armelle , Quertainmont Renaud , Salmon Isabelle , Sansonetti Philippe , Allaoui Abdelmounaaïm
Published: April 2014

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE50-IL-10, IpaH60-IL-10, and IpaH60-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE50-IL-10, IpaH60-IL-10, and IpaH60-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-α and IL-1β mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.
Analysis of HLA-G in Maternal Plasma, Follicular Fluid, and Preimplantation Embryos Reveal an Asymmetric Pattern of Expression
Authors: Shaikly Valerie R. , Morrison Ian E. G. , Taranissi Mohamed , Noble Clare V. , Withey Anna D. , Cherry Richard J. , Blois Sandra M. , Fernández Nelson
Published: April 2014

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.
Modulation of Dendritic Cell Function by Leishmania Parasites
Authors: Soong Lynn
Published: April 2014

The interactions between Leishmania parasites and dendritic cells (DCs) are complex and involve paradoxical functions that can stimulate or halt T cell responses, leading to the control of infection or progression of disease. The magnitude and profile of DC activation vary greatly, depending upon the Leishmania species/strains, developmental stages, DC subsets, serum opsonization, and exogenous DC stimuli involved in the study. In general, the uptake of Leishmania parasites alone can trigger relatively weak and transient DC activation; however, the intracellular parasites (amastigotes) are capable of down-modulating LPS/IFN-γ-stimulated DC activation via multiple mechanisms. This review will highlight current data regarding the initial interaction of DC subsets with invading parasites, the alterations of DC signaling pathways and function by amastigotes, and the impact of DC functions on protective immunity and disease pathogenesis. Available information provides insight into the mechanisms by which DCs discriminate between the types of pathogens and regulate appropriate immune responses.
Cutting Edge: Langerin+ Dendritic Cells in the Mesenteric Lymph Node Set the Stage for Skin and Gut Immune System Cross-Talk
Authors: Chang Sun-Young , Cha Hye-Ran , Igarashi Osamu , Rennert Paul D. , Kissenpfennig Adrien , Malissen Bernard , Nanno Masanobu , Kiyono Hiroshi , Kweon Mi-Na
Published: April 2014

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer’s patch-null mice but abolished in the Peyer’s patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin+CD8α− dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin+ cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin+ dendritic cells in the MLN.
Cutting Edge: Autoimmune Disease in Day 3 Thymectomized Mice Is Actively Controlled by Endogenous Disease-Specific Regulatory T Cells
Authors: Samy Eileen T. , Wheeler Karen M. , Roper Randall J. , Teuscher Cory , Tung Kenneth S. K.
Published: April 2014

Female B6AF1 mice thymectomized on day 3 (d3tx) develop autoimmune ovarian disease (AOD) and dacryoadenitis. It has been hypothesized that d3tx breaks tolerance by depleting late ontogeny regulatory T cells (Treg). We now report that Treg greatly expand over effector T cells in d3tx mice and adoptively suppress autoimmune disease in d3tx recipients. In the d3tx donors, Treg from ovarian lymph nodes (LN) preferentially suppress AOD and Treg from lacrimal gland LN preferentially suppress dacryoadenitis, suggesting they are strategically positioned for disease control. Indeed, the autologous disease in d3tx mice is dramatically enhanced by in vivo depletion of endogenous Treg. Moreover, normal 3-day-old mice possess Treg that suppress AOD and autoimmune gastritis as efficiently as adult cells. Thus, d3tx mice possess disease-relevant Treg of presumed neonatal origin. They accumulate in the regional LN and actively inhibit concurrent autoimmune disease; however, they cannot fully prevent autoimmune disease development.
Dendritic Cell Differentiation State and Their Interaction with NKT Cells Determine Th1/Th2 Differentiation in the Murine Model ofLeishmania majorInfection
Authors: Wiethe Carsten , Debus Andrea , Mohrs Markus , Steinkasserer Alexander , Lutz Manfred , Gessner André
Published: April 2014

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4+ T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Rα signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-α or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.
CD40 on APCs Is Needed for Optimal Programming, Maintenance, and Recall of CD8+ T Cell Memory Even in the Absence of CD4+ T Cell Help
Authors: Hernandez Maria Genevive H. , Shen Lianjun , Rock Kenneth L.
Published: April 2014

CD40 stimulation is one of the many signals that can activate APCs and we have recently shown it to have a unique function in generating maximum primary CD8+ T cell responses. However, whether CD40 signaling plays a role in memory CD8+ T cell responses is still not completely understood. In this study, we show that in the absence of CD40 on all APCs or specifically on dendritic cells, memory CD8+ T cells are generated but at significantly reduced levels. This reduction is due to a contribution of CD40 at several different steps in the generation of CD8+ memory. In the initial T cell response, CD40 contributes to maximizing not only the number of effector cells that are generated but also the programming of ones that will differentiate into memory. Subsequently, CD40 is needed to maintain maximal numbers of the committed memory cells in a manner that is independent of the immunizing Ag. Finally, when memory CD8+ T cells are reactivated there is a variable requirement for CD40 depending on whether CD40 or CD4+ Th cells were present during the primary response. Therefore, CD40 signaling on APCs plays an important role in all phases of a memory CD8+ T cell response.
IL-15 Transpresentation Augments CD8+ T Cell Activation and Is Required for Optimal Recall Responses by Central Memory CD8+ T Cells
Authors: Kokaji Andy I. , Hockley Deanna L. , Kane Kevin P.
Published: April 2014

Although the adaptive immune system has a remarkable ability to mount rapid recall responses to previously encountered pathogens, the cellular and molecular signals necessary for memory CD8+ T cell reactivation are poorly defined. IL-15 plays a critical role in memory CD8+ T cell survival; however, whether IL-15 is also involved in memory CD8+ T cell reactivation is presently unclear. Using artificial Ag-presenting surfaces prepared on cell-sized microspheres, we specifically addressed the role of IL-15 transpresentation on mouse CD8+ T cell activation in the complete absence of additional stimulatory signals. In this study we demonstrate that transpresented IL-15 is significantly more effective than soluble IL-15 in augmenting anti-CD3ε-induced proliferation and effector molecule expression by CD8+ T cells. Importantly, IL-15 transpresentation and TCR ligation by anti-CD3ε or peptide MHC complexes exhibited synergism in stimulating CD8+ T cell responses. In agreement with previous studies, we found that transpresented IL-15 preferentially stimulated memory phenotype CD8+ T cells; however, in pursuing this further, we found that central memory (TCM) and effector memory (TEM) CD8+ T cells responded differentially to transpresented IL-15. TCM CD8+ T cells undergo Ag-independent proliferation in response to transpresented IL-15 alone, whereas TEM CD8+ T cells are relatively unresponsive to transpresented IL-15. Furthermore, upon Ag-specific stimulation, TCM CD8+ T cell responses are enhanced by IL-15 transpresentation, whereas TEM CD8+ T cell responses are only slightly affected, both in vitro and in vivo. Thus, our findings distinguish the role of IL-15 transpresentation in the stimulation of distinct memory CD8+ T cell subsets, and they also have implications for ex vivo reactivation and expansion of Ag-experienced CD8+ T cells for immunotherapeutic approaches.
Lack of TIM-3 Immunoregulation in Multiple Sclerosis
Authors: Yang Li , Anderson David E. , Kuchroo Juhi , Hafler David A.
Published: April 2014

Multiple sclerosis (MS) is an inflammatory disease of the CNS white matter associated with T cell infiltrates and alterations of immune functions that can be measured in the peripheral immune system. TIM-3 has been identified as a central regulator of IFN-γ-secreting type 1 Th (Th1) cells and immune tolerance. In this study, using a newly generated mAb against human TIM-3, we examined TIM-3 function on ex vivo CD4+ T cells isolated from the circulation of healthy subjects and patients with MS. Blocking TIM-3 during T cell stimulation significantly enhanced IFN-γ secretion in control subjects but had no effect in untreated patients with MS, demonstrating a defect in TIM-3 immunoregulation. Treatment with glatiramer acetate or IFN-β reversed this functional defect. Reduced levels and altered kinetics of T cell TIM-3 expression, which was restored in treated patients, is one mechanism that can explain the loss of TIM-3 regulation of T cell function in untreated patients with MS. These data provide functional, mechanistic data for dysregulated TIM-3 immunoregulation in a human autoimmune disease and suggest that approved therapies for the treatment of MS may function in part by restoring TIM-3 immunoregulation of T cell function.