bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Characterization of the CCL25 Promoter in Small Intestinal Epithelial Cells Suggests a Regulatory Role for Caudal-Related Homeobox (Cdx) Transcription Factors
Authors: Ericsson Anna , Kotarsky Knut , Svensson Marcus , Sigvardsson Mikael , Agace William
Published: April 2014

The chemokine CCL25 is selectively and constitutively expressed in the small intestinal epithelium and plays an important role in mediating lymphocyte recruitment to this site. In this study, we demonstrate that CCL25 expression in murine small intestinal epithelial cells is independent of signaling through the lymphotoxin β receptor and is not enhanced by inflammatory stimuli, pathways involved in driving the expression of most other chemokines. We define a transcriptional start site in the CCL25 gene and a region −141 to −5 proximal of exon 1 that is required for minimal promoter activity in the small intestinal epithelial cell lines, MODE-K and mICc12. These cell lines expressed far less CCL25 mRNA than freshly isolated small intestinal epithelial cells indicating that they are missing important factors driving CCL25 expression. The CCL25 promoter contained putative binding sites for the intestinal epithelial-associated Caudal-related homeobox (Cdx) transcription factors Cdx-1 and Cdx-2, and small intestinal epithelial cells but not MODE-K and mICc12 cells expressed Cdx-1 and Cdx-2. EMSA analysis demonstrated that Cdx proteins were present in nuclear extracts from freshly isolated small intestinal epithelial cells but not in MODE-K or mICcl2 cells, and bound to putative Cdx sites within the CCL25 promoter. Finally, cotransfection of MODE-K cells with Cdx transcription factors significantly increased CCL25 promoter activity as well as endogenous CCL25 mRNA levels. Together these results demonstrate a unique pattern of regulation for CCL25 and suggest a role for Cdx proteins in regulating CCL25 transcription.
Modification of MyD88 mRNA Splicing and Inhibition of IL-1β Signaling in Cell Culture and in Mice with a 2′-O-Methoxyethyl-Modified Oligonucleotide
Authors: Vickers Timothy A. , Zhang Hong , Graham Mark J. , Lemonidis Kristina M. , Zhao Chenguang , Dean Nicholas M.
Published: April 2014

A number of proinflammatory cytokines, including IL-1β, signal through the adaptor protein MyD88. This signaling leads to phosphorylation of IL-1R-associated kinase-1 (IRAK-1) and, ultimately, activation of the NF-κB transcription factor. A splice variant of MyD88 (MyD88S), which lacks the ability to couple IRAK-1 to NF-κB, has been described. A chemically modified antisense oligonucleotide (ASO) that alters the splicing ratio of MyD88 to MyD88S in both cell culture and in animals has been identified. The ASO (ISIS 337846) binds to exon II donor sites in the MyD88 pre-mRNA. By manipulating levels of MyD88 splicing, proinflammatory signaling through the IL-1R has been shown to be diminished, both in cell culture and in mouse liver. To our knowledge, this represents the first example of modulation of RNA splicing of an endogenous gene target in animals after systemic ASO dosing and suggests that this mechanism may be useful as a novel modulator of inflammatory stimuli.
Genetic Dissection of Vasculitis, Myeloperoxidase-Specific Antineutrophil Cytoplasmic Autoantibody Production, and Related Traits in Spontaneous Crescentic Glomerulonephritis-Forming/Kinjoh Mice
Authors: Hamano Yoshitomo , Tsukamoto Kazuyuki , Abe Masaaki , Sun Guo Dong , Zhang Danqing , Fujii Hiroaki , Matsuoka Shuji , Tanaka Masumi , Ishida-Okawara Akiko , Tachikawa Hitoshi , Nishimura Hiroyuki , Tokunaka Kazuhiro , Hirose Sachiko , Suzuki Kazuo
Published: April 2014

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B6× SCG/Kj) F2 intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B6 on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.
Ancestral Organization of the MHC Revealed in the AmphibianXenopus
Authors: Ohta Yuko , Goetz Wilfried , Hossain M. Zulfiquer , Nonaka Masaru , Flajnik Martin F.
Published: April 2014

With the advent of the Xenopus tropicalis genome project, we analyzed scaffolds containing MHC genes. On eight scaffolds encompassing 3.65 Mbp, 122 MHC genes were found of which 110 genes were annotated. Expressed sequence tag database screening showed that most of these genes are expressed. In the extended class II and class III regions the genomic organization, excluding several block inversions, is remarkably similar to that of the human MHC. Genes in the human extended class I region are also well conserved in Xenopus, excluding the class I genes themselves. As expected from previous work on the Xenopus MHC, the single classical class I gene is tightly linked to immunoproteasome and transporter genes, defining the true class I region, present in all nonmammalian jawed vertebrates studied to date. Surprisingly, the immunoproteasome gene PSMB10 is found in the class III region rather than in the class I region, likely reflecting the ancestral condition. Xenopus DMα, DMβ, and C2 genes were identified, which are not present or not clearly identifiable in the genomes of any teleosts. Of great interest are novel V-type Ig superfamily (Igsf) genes in the class III region, some of which have inhibitory motifs (ITIM) in their cytoplasmic domains. Our analysis indicates that the vertebrate MHC experienced a vigorous rearrangement in the bony fish and bird lineages, and a translocation and expansion of the class I genes in the mammalian lineage. Thus, the amphibian MHC is the most evolutionary conserved MHC so far analyzed.
Phospholipase D1 Regulates Phagocyte Adhesion
Authors: Iyer Shankar S. , Agrawal Reitu S. , Thompson Christopher R. , Thompson Steven , Barton James A. , Kusner David J.
Published: April 2014

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation, transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Because phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen, or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy indicated that PLD1 exhibited partial colocalization with actin filaments at the adherent interface, in proximity to the focal adhesion protein, paxillin. Reductions in PLD activity by chemical inhibitors or specific short-interfering RNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion and was accompanied by reductions in total cellular F-actin. These data support the hypotheses that adhesion stimulates PLD activity, and that PLD1 regulates the initial stages of phagocyte adhesion. Stimulation of PLD activity may promote adhesion-dependent phagocyte effector responses.
The Combined CTA1-DD/ISCOM Adjuvant Vector Promotes Priming of Mucosal and Systemic Immunity to Incorporated Antigens by Specific Targeting of B Cells
Authors: Helgeby Anja , Robson Neil C. , Donachie Anne M. , Beackock-Sharp Helen , Lövgren Karin , Schön Karin , Mowat Allan , Lycke Nils Y.
Published: April 2014

The cholera toxin A1 (CTA1)-DD/QuilA-containing, immune-stimulating complex (ISCOM) vector is a rationally designed mucosal adjuvant that greatly potentiates humoral and cellular immune responses. It was developed to incorporate the distinctive properties of either adjuvant alone in a combination that exerted additive enhancing effects on mucosal immune responses. In this study we demonstrate that CTA1-DD and an unrelated Ag can be incorporated together into the ISCOM, resulting in greatly augmented immunogenicity of the Ag. To demonstrate its relevance for protection against infectious diseases, we tested the vector incorporating PR8 Ag from the influenza virus. After intranasal immunization we found that the immunogenicity of the PR8 proteins were significantly augmented by a mechanism that was enzyme dependent, because the presence of the enzymatically inactive CTA1R7K-DD mutant largely failed to enhance the response over that seen with ISCOMs alone. The combined vector was a highly effective enhancer of a broad range of immune responses, including specific serum Abs and balanced Th1 and Th2 CD4+ T cell priming as well as a strong mucosal IgA response. Unlike unmodified ISCOMs, Ag incorporated into the combined vector could be presented by B cells in vitro and in vivo as well as by dendritic cells; it also accumulated in B cell follicles of draining lymph nodes when given s.c. and stimulated much enhanced germinal center reactions. Strikingly, the enhanced adjuvant activity of the combined vector was absent in B cell-deficient mice, supporting the idea that B cells are important for the adjuvant effects of the combined CTA1-DD/ISCOM vector.
A Mechanism of Virulence: Virulent Mycobacterium tuberculosis Strain H37Rv, but Not Attenuated H37Ra, Causes Significant Mitochondrial Inner Membrane Disruption in Macrophages Leading to Necrosis
Authors: Chen Minjian , Gan Huixian , Remold Heinz G.
Published: April 2014

Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48–72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca2+ flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Δψm) loss caused by mitochondrial permeability transition. Dissipation of Δψm also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca2+ loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Δψm 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.
Dectin-1 and TLRs Permit Macrophages to Distinguish between Different Aspergillus fumigatus Cellular States
Authors: Gersuk Geoffrey M. , Underhill David M. , Zhu Liqun , Marr Kieren A.
Published: April 2014

Aspergillus fumigatus is a common cause of invasive and allergic pulmonary disease. Resting conidia of the filamentous fungus are constantly inhaled, but cause infection only after initiating hyphal growth. In this study, we have explored whether macrophages can distinguish between resting spores and the maturing, potentially invasive form of the fungus. Although macrophages bind and ingest A. fumigatus resting conidia efficiently, there is little inflammatory response; NF-κβ is not activated, inflammatory cytokines are not induced, and reactive oxygen species are not produced. However, maturing A. fumigatus conidia and germ tubes stimulate NF-κβ, secretion of proinflammatory cytokines and production of reactive oxygen by human monocyte-derived macrophages and murine macrophages from multiple anatomical sites. These responses are in part mediated by dectin-1, which binds cell wall β-glucan that is not present on the surface of dormant conidia, but is present after cellular swelling and loss of the hydrophobic proteinaceous cell wall. Dectin-1 binding to germ tubes augments, but is not required for, TLR2-mediated inflammatory cytokine secretion. Dectin-1 recognition of germ tubes also stimulates TNF-α production in the absence of both TLR2 and MyD88 signaling. These data demonstrate one mechanism by which the pulmonary inflammatory response is tailored toward metabolically active cells, thereby avoiding unnecessary tissue damage with frequent inhalation of ubiquitous spores.
Structure and Regulatory Profile of the Monkeypox Inhibitor of Complement: Comparison to Homologs in Vaccinia and Variola and Evidence for Dimer Formation
Authors: Liszewski M. Kathryn , Leung Marilyn K. , Hauhart Richard , Buller R. Mark L. , Bertram Paula , Wang Xuefeng , Rosengard Ariella M. , Kotwal Girish J. , Atkinson John P.
Published: April 2014

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5–30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were ∼100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.
Matrix Metalloproteinase-9 Deficiency Impairs Host Defense against Abdominal Sepsis
Authors: Renckens Rosemarijn , Roelofs Joris J. T. H. , Florquin Sandrine , de Vos Alex F. , Lijnen H. Roger , van’t Veer Cornelis , van der Poll Tom
Published: April 2014

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9−/−) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9−/− neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9−/− mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9−/− mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9−/− mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9−/− mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.
Modulation of Neonatal Microbial Recognition: TLR-Mediated Innate Immune Responses Are Specifically and Differentially Modulated by Human Milk
Authors: LeBouder Emmanuel , Rey-Nores Julia E. , Raby Anne-Catherine , Affolter Michael , Vidal Karine , Thornton Catherine A. , Labéta Mario O.
Published: April 2014

The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1–5) of breast milk on ligand-induced cell stimulation via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS. Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation, suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including those coding for signal intermediates and regulators. A proteinaceous milk component of ≥80 kDa was found to be responsible for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms regulating TLR activation.
Epitope Enhancement of a CD4 HIV Epitope toward the Development of the Next Generation HIV Vaccine
Authors: Okazaki Takahiro , Pendleton C. David , Sarobe Pablo , Thomas Elaine K. , Iyengar Sujatha , Harro Clayton , Schwartz David , Berzofsky Jay A.
Published: April 2014

Virus-specific CD4+ T cell help and CD8+ cytotoxic T cell responses are critical for maintenance of effective immunity in chronic viral infections. The importance of CD4+ T cells has been documented in HIV infection. To investigate whether a stronger CD4+ T cell response can be induced by modifications to enhance the T1 epitope, the first CD4+ T cell epitope discovered in HIV-1-gp120, we developed a T1-specific CD4+ T cell line from a healthy volunteer immunized with a canarypox vector expressing gp120 and boosted with recombinant gp120. This T1-specific CD4+ T cell line was restricted to DR13, which is common in U.S. Caucasians and African-Americans and very frequent in Africans. Peptides with certain amino acid substitutions in key positions induced enhanced specific CD4+ T cell proliferative responses at lower peptide concentration than the original epitope. This relatively conserved CD4 epitope improved by the epitope enhancement strategy could be a component of a more effective second generation vaccine construct for HIV infection.
Genome-Wide Analysis Reveals a Highly Diverse CD8 T Cell Response to Murine Cytomegalovirus
Authors: Munks Michael W. , Gold Marielle C. , Zajac Allison L. , Doom Carmen M. , Morello Christopher S. , Spector Deborah H. , Hill Ann B.
Published: April 2014

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2b-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.
A Quantitative Trait Loci Analysis to Map Genes Involved in Lipopolysaccharide-Induced Inflammatory Response: Identification of Macrophage Scavenger Receptor 1 as a Candidate Gene
Authors: Fulton William B. , Reeves Roger H. , Takeya Motohiro , De Maio Antonio
Published: April 2014

Septic shock, which is a major complication observed after trauma and other human diseases, is likely the product of a prolonged and poorly controlled systemic inflammatory response. Symptoms of sepsis can be partially reproduced by injection of bacterial LPS in mice. Differences in mortality between C57BL/6Jhigh and A/Jlow mice after LPS injection have been previously observed and correlated with differences in the inflammatory response between these two inbred strains. In the present study, we have mapped four loci responsible for differences in levels of LPS-induced IL-10, named modifier of IL-10, between the two strains. A locus within mouse chromosome 8 was confirmed using chromosome 8 consomic mice. This locus was further reduced in size by haplotype analysis and evaluated by the presence of potential candidate genes. The macrophage scavenger receptor 1 (Msr1) within this locus emerged as a candidate gene based on differences at the expression and structural levels between C57BL/6J and A/J mice. In comparison with wild-type (C57BL/6J) mice, Msr1 knockout mice displayed reduced levels of LPS-induced IL-10, but not of TNF-α or IL-6, confirming a specific role for this gene in the regulation of IL-10. These results suggest that Msr1 is involved in the regulation of the anti-inflammatory process, thus offering a new perspective on the molecular mechanisms involved in endotoxemia and sepsis.
Pancreatitis-Associated Protein I Suppresses NF-κB Activation through a JAK/STAT-Mediated Mechanism in Epithelial Cells
Authors: Folch-Puy Emma , Granell Susana , Dagorn Jean C. , Iovanna Juan L. , Closa Daniel
Published: April 2014

Pancreatitis-associated protein I (PAP I), also known as HIP, p23, or Reg2 protein, has recently been implicated in the endogenous regulation of inflammation. Although it was initially characterized as a protein that is overexpressed in acute pancreatitis, PAP I has also been associated with a number of inflammatory diseases, such as Crohn’s disease. Knowing that PAP I and IL-10 responses share several features, we have used a pancreatic acinar cell line (AR42J) to assess the extent to which their expression is reciprocally regulated, and whether the JAK/STAT and NF-κB signaling pathways are involved in the suppression of inflammation mediated by PAP I. We observed that PAP I is induced in epithelial cells by IL-10 and by PAP I itself. In contrast, we found phosphorylation and nuclear translocation of STAT3 and induction of suppressor of cytokine signaling 3 in response to PAP I exposure. Finally, a JAK-specific inhibitor, tyrphostin AG490, markedly prevented PAP I-induced NF-κB inhibition, pointing to a cross-talk between JAK/STAT3 and NF-κB signaling pathways. Together, these findings indicate that PAP I inhibits the inflammatory response by blocking NF-κB activation through a STAT3-dependent mechanism. Important functional similarities to the anti-inflammatory cytokine IL-10 suggest that PAP I could play a role similar to that of IL-10 in epithelial cells.
Regulation of IL-1 Family Cytokines IL-1α, IL-1 Receptor Antagonist, and IL-18 by 1,25-Dihydroxyvitamin D3 in Primary Keratinocytes
Authors: Kong Juan , Grando Sergei A. , Li Yan Chun
Published: April 2014

IL-1 family cytokines are key mediators of inflammatory response. Excessive production of these cytokines by keratinocytes has been implicated in inflammatory and hyperproliferative skin diseases. Given the immunosuppressive role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its clinical application in treatment of psoriasis, we investigated the effect of 1,25(OH)2D3 on the expression of IL-1α, intracellular IL-1 receptor antagonist (icIL-1Ra), and IL-18 in mouse primary keratinocytes. Treatment of keratinocytes with 1,25(OH)2D3 increased the expression of IL-1α and icIL-1Ra and decreased the expression of IL-18 in dose- and time-dependent manners. The magnitude of icIL-1Ra induction was much greater than that of IL-1α so that the ratio of icIL-1Ra to IL-1α was markedly increased, leading to repression of IL-1 activity. The regulation of these three cytokines by 1,25(OH)2D3 was mediated by vitamin D receptor (VDR), as 1,25(OH)2D3 had no effect in VDR−/− keratinocytes, whereas the effect was restored in cells derived from VDR−/− mice expressing human VDR. 1,25(OH)2D3 appeared to use different mechanisms to regulate the biosynthesis of IL-1α and icIL-1Ra: it increased IL-1α mRNA stability whereas it enhanced icIL-1Ra gene transcription. The basal IL-18 expression and activity were much higher in VDR−/− keratinocytes and skin, underscoring the importance of the repressive role of vitamin D in IL-18 production. Similar regulation of these cytokines was also seen in primary human keratinocytes. Collectively, these results suggest that vitamin D modulates cutaneous inflammatory reactions, at least in part, by increasing the IL-1Ra to IL-1α ratio and suppressing IL-18 synthesis in keratinocytes.
Mast Cell β-Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities
Authors: Pang Linhua , Nie Mei , Corbett Lisa , Sutcliffe Amy , Knox Alan J.
Published: April 2014

Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by β-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of β-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified β-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human β-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. β-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with β-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of β-tryptase on RANTES. These findings suggest that mast cell β-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.
MyD88 and TNF Receptor-Associated Factor 6 Are Critical Signal Transducers in Helicobacter pylori-Infected Human Epithelial Cells
Authors: Hirata Yoshihiro , Ohmae Tomoya , Shibata Wataru , Maeda Shin , Ogura Keiji , Yoshida Haruhiko , Kawabe Takao , Omata Masao
Published: April 2014

Helicobacter pylori induces NF-κB activation, leading to mucosal inflammation via cag pathogenicity island. Although recent studies have implicated several candidate proteins of both H. pylori and host, the molecular mechanism by which H. pylori activates NF-κB remains unclear. The aim of this study was to analyze the mechanism of cag pathogenicity island-mediated NF-κB activation in epithelial cells. The responses of human cell lines and mouse embryonic fibroblasts to infection with wild-type H. pylori or cagE mutant were investigated. The effect of small interfering RNAs (siRNAs) for several NF-κB signaling intermediate molecules was evaluated in H. pylori-induced IκBα phosphorylation and IL-8 production. Protein interactions of exogenously expressed TNFR-associated factor 6 (TRAF6) and MyD88 or receptor-interacting protein 2 and nucleotide-binding oligomerization domain 1 or those of endogenous IκB kinase, TGF-β-activated kinase 1 (TAK1), and TRAF6 were assessed by immunoprecipitation. Cag pathogenicity island-dependent NF-κB activation was observed in human cell lines, but not in mouse fibroblasts. In human epithelial cells, H. pylori-induced IκBα phosphorylation and IL-8 production were severely inhibited by siRNAs directed against TAK1, TRAF6, and MyD88. In contrast, siRNAs for TRAF2, IL-1R-associated kinases 1 and 4, and cell surface receptor proteins did not affect these responses. H. pylori infection greatly enhanced MyD88 and TRAF6 complex formation in a cag-dependent manner, but did not enhance Nod1 and receptor-interacting protein 2 complex formation. H. pylori also induced TAK1 and TRAF6 complexes. These results suggest that the cag pathogenicity island of H. pylori is a cell type-specific NF-κB activator. TAK1, TRAF6, and MyD88 are important signal transducers in H. pylori-infected human epithelial cells.
Microglia Recognize Double-Stranded RNA via TLR3
Authors: Town Terrence , Jeng David , Alexopoulou Lena , Tan Jun , Flavell Richard A.
Published: April 2014

Microglia are CNS resident innate immune cells of myeloid origin that become activated and produce innate proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors for pathogen-associated molecular patterns that drive innate immune responses. We have recently shown that mice deficient in TLR3 (TLR3−/− mice) are resistant to lethal encephalitis and have reduced microglial activation after infection with West Nile virus, a retrovirus that produces dsRNA. We wished to determine whether microglia recognize dsRNA through the TLR3 pathway. In vitro, murine wild-type primary cultured microglia responded to synthetic dsRNA polyinosinic-polycytidylic acid (poly(I:C)) by increasing TLR3 and IFN-β mRNA and by morphologic activation. Furthermore, wild-type microglia dose dependently secreted TNF-α and IL-6 after poly(I:C) challenge, whereas TLR3−/− microglia produced diminished cytokines. Activation of MAPK occurred in a time-dependent fashion following poly(I:C) treatment of wild-type microglia, but happened with delayed kinetics in TLR3−/− microglia. As an in vivo model of encephalitis, wild-type or TLR3−/− mice were injected intracerebroventricularly with poly(I:C) or LPS, and microglial activation was assessed by cell surface marker or phospho-MAPK immunofluorescence. After intracerebroventricular injection of poly(I:C), microgliosis was clearly evident in wild-type mice but was nearly absent in TLR3−/− animals. When taken together, our results demonstrate that microglia recognize dsRNA through TLR3 and associated signaling molecules and suggest that these cells are key sensors of dsRNA-producing viruses that may invade the CNS.
Preprotachykinin-A Gene Products Are Key Mediators of Lung Injury in Polymicrobial Sepsis
Authors: Puneet Padmam , Hegde Akhil , Ng Siaw Wei , Lau Hon Yen , Lu Jia , Moochhala Shabbir M. , Bhatia Madhav
Published: April 2014

Preprotachykinin-A (PPT-A) gene products substance P and neurokinin-A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT-A gene products in lung injury in sepsis, polymicrobial sepsis was induced by cecal ligation and puncture in PPT-A gene-deficient mice (PPT-A−/−) and the wild-type control mice (PPT-A+/+). PPT-A gene deletion significantly protected against mortality, delayed the onset of lethality, and improved the long-term survival following cecal ligation and puncture-induced sepsis. PPT-A−/− mice also had significantly attenuated inflammation and damage in the lungs. The data suggest that deletion of the PPT-A gene may have contributed to the disruption in recruitment of inflammatory cells resulting in protection against tissue damage, as in these mice the sepsis-associated increase in chemokine levels is significantly attenuated.
T Cell Responses in Dengue Hemorrhagic Fever: Are Cross-Reactive T Cells Suboptimal?
Authors: Mongkolsapaya Juthathip , Duangchinda Thaneeya , Dejnirattisai Wanwisa , Vasanawathana Sirijit , Avirutnan Panisadee , Jairungsri Aroonroong , Khemnu Nuanpan , Tangthawornchaikul Nattaya , Chotiyarnwong Pojchong , Sae-Jang Kanokwan , Koch Michael , Jones Yvonne , McMichael Andrew , Xu Xiaoning , Malasit Prida , Screaton Gavin
Published: April 2014

Dengue virus infection poses a growing public health and economic burden in a number of tropical and subtropical countries. Dengue circulates as a number of quasispecies, which can be divided by serology into four groups or serotypes. An interesting feature of Dengue, recognized over five decades ago, is that most severe cases that show hemorrhagic fever are not suffering from a primary infection. Instead, they are reinfected with a virus of different serotype. This observation poses considerable problems in vaccine design, and it is therefore imperative to gain a full understanding of the mechanisms underlying this immunological enhancement of disease. In this study, we examined a T cell epitope restricted by HLA-A*24, a major MHC class I allele, in Southeast Asia in a cohort of children admitted to a hospital with acute Dengue infection. The cytokine profiles and the degranulation capacity of T cells generated to this epitope are defined and compared across different viral serotypes. Cross-reactive Dengue-specific T cells seem to show suboptimal degranulation but high cytokine production, which may contribute to the development of the vascular leak characteristic of Dengue hemorrhagic fever.
Sensitization of IL-2 Signaling through TLR-7 Enhances B Lymphoma Cell Immunogenicity
Authors: Tomic Jelena , White Dionne , Shi Yonghong , Mena Jenny , Hammond Caitlin , He Liwei , Miller Richard L. , Spaner David E.
Published: April 2014

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs, the effects of IL-2 and the TLR-7 agonist, S28690, on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members, production of TNF-α and IL-10, and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However, IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C, which inhibited tumor cell proliferation, “switched off” IL-10 production, and caused essentially all CLL cells (regardless of clinical stage) to acquire a CD83highCD80highCD86highCD54high surface phenotype marked by the activation of STAT-1 without STAT-3. These findings suggest that TLR-7 “licenses” human B cells to respond to cytokines of the adaptive immune system (such as IL-2) and provide a strategy to increase the immunogenicity of lymphoma cells for therapeutic purposes.
Injection heights of springtime biomass-burning plumes over peninsular Southeast Asia and their impacts on long-range pollutant transport
Authors: Jian Y. , Fu T.-M.
Published: April 2014

Abstract. We analyzed observations from the Multi-angle Imaging SpectroRadiometer (MISR) to determine the injection heights of biomass-burning smoke plumes over peninsular Southeast Asia (PSEA, here defined as Vietnam, Cambodia, Thailand, Laos, and Myanmar) in the spring, with the goal of evaluating the impacts on long-range pollutant transport. We retrieved the heights of 22 000 MISR smoke pixels from 607 smoke plumes over PSEA during February to April of the years 2001–2010. Forty-five percent of the analyzed smoke pixels were above the local mean boundary layer (1 km) at MISR overpass time (10:30 a.m. local time). We used the GEOS–Chem model to simulate the transport of PSEA biomass-burning pollutants in March 2001. On a monthly mean basis, we found that the direct injection of 40% of the PSEA biomass-burning emissions had little impact on the long-range transport of CO to downwind regions, compared to a control simulation where all biomass-burning emissions were released in the boundary layer. This was because CO at the surface over PSEA was efficiently lifted into the free troposphere by deep convection associated with synoptic-scale weather systems. For pollutants with lifetimes shorter than the synoptic timescale, such as black carbon aerosol (BC), their long-range transport was much more sensitive to the initial plume injection height. The direct injection of NOx from PSEA biomass burning into the free troposphere drove increased formation and transport of peroxyacetyl nitrate (PAN), which in turn led to a small increase in ozone over downwind southern China and the northwestern Pacific. The Pacific subtropical high transported BC emitted from PSEA biomass burning to the marine boundary layer over the tropical northwestern Pacific. We compared our model results to aircraft measurements over the northwestern Pacific during the TRACE-P campaign (March 2001). The direct injection of 40% of the PSEA biomass-burning pollutants into the free troposphere in the model led to a more pronounced BC peak at 3 km over the northwestern Pacific. Our analysis highlights the point that the injection heights of smoke plumes presents great uncertainty over the interpretation of BC measurements downwind of biomass-burning regions.
A multi-year methane inversion using SCIAMACHY, accounting for systematic errors using TCCON measurements
Authors: Houweling S. , Krol M. , Bergamaschi P. , Frankenberg C. , Dlugokencky E. J. , Morino I. , Notholt J. , Sherlock V. , Wunch D. , Beck V. , Gerbig C. , Chen H. , Kort E. A. , Röckmann T. , Aben I.
Published: April 2014

Abstract. This study investigates the use of total column CH4 (XCH4) retrievals from the SCIAMACHY satellite instrument for quantifying large-scale emissions of methane. A unique data set from SCIAMACHY is available spanning almost a decade of measurements, covering a period when the global CH4 growth rate showed a marked transition from stable to increasing mixing ratios. The TM5 4DVAR inverse modelling system has been used to infer CH4 emissions from a combination of satellite and surface measurements for the period 2003–2010. In contrast to earlier inverse modelling studies, the SCIAMACHY retrievals have been corrected for systematic errors using the TCCON network of ground-based Fourier transform spectrometers. The aim is to further investigate the role of bias correction of satellite data in inversions. Methods for bias correction are discussed, and the sensitivity of the optimized emissions to alternative bias correction functions is quantified. It is found that the use of SCIAMACHY retrievals in TM5 4DVAR increases the estimated inter-annual variability of large-scale fluxes by 22% compared with the use of only surface observations. The difference in global methane emissions between 2-year periods before and after July 2006 is estimated at 27–35 Tg yr−1. The use of SCIAMACHY retrievals causes a shift in the emissions from the extra-tropics to the tropics of 50 ± 25 Tg yr−1. The large uncertainty in this value arises from the uncertainty in the bias correction functions. Using measurements from the HIPPO and BARCA aircraft campaigns, we show that systematic errors in the SCIAMACHY measurements are a main factor limiting the performance of the inversions. To further constrain tropical emissions of methane using current and future satellite missions, extended validation capabilities in the tropics are of critical importance.
Is tropospheric ozone over southern Africa really increasing? Evidence from sonde and aircraft profiles
Authors: Thompson A. M. , Balashov N. V. , Witte J. C. , Coetzee J. G. R. , Thouret V. , Posny F.
Published: April 2014

Abstract. Ozonesonde records from the early 1990's through 2008 over two subtropical stations, Irene (near Pretoria, South Africa) and Réunion Island (21° S, 55° W, ~3500 km NE of Irene in the Indian Ocean) were reported to exhibit free tropospheric (FT) ozone increases. Over Irene a large increase in the urban-influenced boundary layer (BL, 1.5–4 km) was also observed during the 18 year period, equivalent to 30% decade−1. Here we show that the Irene BL trend is at least partly due to a gradual change in the sonde launch times from early morning to the midday period. The FT ozone profiles over Irene in 1990–2007 are re-examined, filling in a 1995–1999 gap with ozone profiles taken by Measurements of Ozone by Airbus In-service Aircraft (MOZAIC) over nearby Johannesburg. A multivariate regression model that accounts for the annual ozone cycle, ENSO and possible tropopause changes was applied to monthly averaged Irene data from 4–11 km and to 1992–2011 Réunion sonde data from 4–15 km. Statistically significant trends appear predominantly in the middle and upper troposphere (UT, 4–11 km over Irene, 5–13 km over Réunion) in winter (June–August), with increases ~1 ppbv yr−1 over Irene and ~2 ppbv yr−1 over Réunion. These changes are equivalent to ~25% and 40–50% decade−1, respectively. Both stations also display smaller positive trends in summer with a 50% decade−1 ozone increase near the tropopause over Réunion in December. To explain the ozone increases, we investigated a time series of dynamical markers, e.g., potential vorticity (PV) at 330–350 K. PV affects UT ozone over Irene in November–December but displays little relationship to ozone over Réunion. A more likely reason for wintertime FT ozone increases over Irene and Réunion appears to be long-range transport of growing pollution in the Southern Hemisphere. The ozone increases are consistent with trajectory origins of air parcels sampled by the sondes and with recent NOx emissions trends estimated for Africa, South America, and Madagascar. For Réunion trajectories also point to pollution sources from the eastern Indian Ocean.
Thin terrestrial sediment deposits on intertidal sandflats: effects on pore-water solutes and juvenile bivalve burial behaviour
Authors: Hohaia A. , Vopel K. , Pilditch C. A.
Published: April 2014

Abstract. Nearshore zones experience increased sedimentation due to coastal development and enhanced loads of fine terrestrial sediment (hereafter, TS) in river waters. Deposition of TS can alter seabed biogeochemical processes but the effects on benthic ecosystem functioning are unknown. The results of a past experiment with defaunated, intertidal sediment suggest that a decrease in the oxygenation of this sediment by a thin (mm) TS deposit causes substrate rejection (refusal to bury) by post-settlement juvenile recruits of the tellinid bivalve Macomona liliana. We further examined this behaviour, asking if such deposits negatively affect burial when applied to intertidal sediment that is oxygenated by bioturbation (C) or depleted of dead and living organic matter (D). We observed recruits on the surface of four treatments: C, D, and the same sediments to which we added a 1.7–1.9 mm layer of TS (CTS, DTS). The TS deposit decreased the oxygenation and the pH of the underlying intertidal sediment (CTS) confirming previous results, but significantly increased but not decreased the probability of burial, irrespectively of treatment. Juveniles more likely buried into C than into D. The mechanism that caused previously observed substrate rejection by post-settlement juvenile M. liliana remains unclear but our results suggest that contact of the recruits with the TS deposit does not cause substrate rejection. We now hypothesise that conditioning of sediment by bioturbation can mediate negative effects of TS deposits on the recruits' burial behaviour.
Low-molecular-weight hydroxyacids in marine atmospheric aerosol: evidence of a marine microbial origin
Authors: Miyazaki Y. , Sawano M. , Kawamura K.
Published: April 2014

Abstract. Lactic acid (LA) and glycolic acid (GA), which are low-molecular-weight hydroxyacids, were identified in the particle and gas phases within the marine atmospheric boundary layer over the western subarctic North Pacific. Major portion of LA (81%) and GA (57%) were present in the particulate phase, which is consistent with the presence of a hydroxyl group in these molecules leading to the low volatility of the compounds. The average concentration of LA in more biologically influenced marine aerosols (average 33 ± 58 ng m−3) was substantially higher than that in less biologically influenced aerosols (average 11 ± 12 ng m−3). Over the oceacnic region of phytoplankton blooms, the concentration of aerosol LA was comparable to that of oxalic acid, which was the most abundant diacid during the study period. A positive correlation was found between the LA concentrations in more biologically influenced aerosols and chlorophyll a in seawater (r2 = 0.56), suggesting an important production of aerosol LA possibly associated with microbial (e.g., lactobacillus) activity in seawater and/or aerosols. Our finding provides a new insight into the poorly quantified microbial sources of marine organic aerosols (OA) because such low-molecular-weight hydroxyacids are key intermediates for OA formation.