bluuebunny/crossref_metadata_2025 huggingface dataset:
E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification
Authors: Yabuki Munehisa , Ordinario Ellen C. , Cummings W. Jason , Fujii Monica M. , Maizels Nancy
Published: April 2014

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged IgλR gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged λR gene, we show that E2A must function in cis to promote diversification and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that λR/E2A colocalizations are most prominent in G1. We further show that expression of the E2A antagonist Id1 prevents λR/E2A colocalizations in G1 and impairs diversification but not transcription of λR. Thus, E2A acts in cis to promote Ig gene diversification, and G1 phase is the critical window for E2A action.
A Simple Mycobacterial Monomycolated Glycerol Lipid Has Potent Immunostimulatory Activity
Authors: Andersen Claire S. , Agger Else Marie , Rosenkrands Ida , Gomes Jessica M. , Bhowruth Veemal , Gibson Kevin J. C. , Petersen Rune V. , Minnikin David E. , Besra Gurdyal S. , Andersen Peter
Published: April 2014

It is a long held belief that the strong immunostimulatory activity of the Mycobacterium bovis bacillus Calmette-Guérin vaccine and Freund’s complete adjuvant is due to specific mycobacterial cell envelope components, such as lipids and polysaccharides. Implicated mycobacterial lipids include, among others, the so-called cord factor or trehalose dimycolate, but limited information is available regarding the precise molecular nature of the stimulatory components responsible for the interaction with human APCs. In this regard, the majority of research aimed at identifying and characterizing individual immunostimulatory mycobacterial lipids has been performed in the murine system using bone marrow-derived dendritic cells. In this study, it is documented that potent immunostimulatory activity lies within the bacillus Calmette-Guérin nonpolar lipid class. This activity can be narrowed down to a remarkably simple monomycolyl glycerol (MMG) with the ability to stimulate human dendritic cells as assessed by enhanced expression of activation markers and the release of proinflammatory cytokines. A synthetic analog of MMG based on 32 carbons (C32) was found to exhibit comparable levels of immunostimulatory activities. Immunization of mice with the tuberculosis vaccine candidate, Ag85B-ESAT-6, in MMG or the synthetic analog using cationic liposomes as the delivery vehicle was found to give rise to a prominent Th1 response characterized by significant levels of IFN-γ. Together, this development opens up the possibility of producing a novel class of chemically defined lipid adjuvants to enhance the activity of new vaccine formulations, directed against infectious agents including tuberculosis.
MicroRNA-221–222 Regulate the Cell Cycle in Mast Cells
Authors: Mayoral Ramon J. , Pipkin Matthew E. , Pachkov Mikhail , van Nimwegen Erik , Rao Anjana , Monticelli Silvia
Published: April 2014

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221–222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221–222 transcription. Overexpression of miR-221–222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221–222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3′ untranslated region, only one of which is subject to miR-221–222 regulation. Additionally, transgenic expression of miR-221–222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221–222 transcriptional regulation as well as evidences that miR-221–222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.
Cutting Edge: Developmental Stage-Specific Recruitment of Cohesin to CTCF Sites throughout Immunoglobulin Loci during B Lymphocyte Development
Authors: Degner Stephanie C. , Wong Timothy P. , Jankevicius Gytis , Feeney Ann J.
Published: April 2014

Contraction of the large Igh and Igκ loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJH or Jκ genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the VH and Vκ regions. However, CTCF enrichment in the Igh locus, but not the Igκ locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.
Human Cytomegalovirus Paralyzes Macrophage Motility through Down-Regulation of Chemokine Receptors, Reorganization of the Cytoskeleton, and Release of Macrophage Migration Inhibitory Factor
Authors: Frascaroli Giada , Varani Stefania , Blankenhorn Nina , Pretsch Robert , Bacher Michael , Leng Lin , Bucala Richard , Landini Maria Paola , Mertens Thomas
Published: April 2014

Macrophages contribute to host defense and to the maintenance of immune homeostasis. Conversely, they are important targets of human cytomegalovirus (HCMV), a herpesvirus that has evolved many strategies to modulate the host immune response. Because an efficient macrophage trafficking is required for triggering an adequate immune response, we investigated the effects exerted by HCMV infection on macrophage migratory properties. By using endotheliotropic strains of HCMV, we obtained high rates of productively infected human monocyte-derived macrophages (MDM). Twenty-four hours after infection, MDM showed reduced polar morphology and became unable to migrate in response to inflammatory and lymphoid chemokines, bacterial products and growth factors, despite being viable and metabolically active. Although chemotactic receptors were only partially affected, HCMV induced a dramatic reorganization of the cytoskeleton characterized by rupture of the microtubular network, stiffness of the actin fibers, and collapse of the podosomes. Furthermore, supernatants harvested from infected MDM contained high amounts of macrophage migration inhibitory factor (MIF) and were capable to block the migration of neighboring uninfected MDM. Because immunodepletion of MIF from the conditioned medium completely restored MDM chemotaxis, we could show for the first time a functional role of MIF as an inhibitor of macrophage migration in the context of HCMV infection. Our findings reveal that HCMV uses different mechanisms to interfere with movement and positioning of macrophages, possibly leading to an impairment of antiviral responses and to an enhancement of the local inflammation.
The Loss and Gain of Marginal Zone and Peritoneal B Cells Is Different in Response to Relapsing Fever and Lyme Disease Borrelia
Authors: Malkiel Susan , Kuhlow Christopher J. , Mena Patricio , Benach Jorge L.
Published: April 2014

T cell-independent Abs are protective against Lyme disease and relapsing fever, illnesses caused by Borrelia spirochetes with distinct blood-borne phases of infection. To understand this protective response, we characterized splenic and peritoneal B cell compartments during infection using flow cytometry and immunohistochemistry. In the spleen, early after infection, Borrelia crocidurae, a relapsing fever species, induced a striking loss of marginal zone (MZ) B cells from the MZ, while Borrelia burgdorferi, the agent of Lyme disease, induced the expansion of this subset. At the same time, no significant changes were observed in follicular B cells in response to either species of Borrelia. In the peritoneal cavity, a further loss was demonstrated early in response to B. crocidurae in the B1b, B1c, and B2 cell subsets, but B1a cells were not significantly altered. The loss of B1c and B2 cells was sustained through subsequent peaks of spirochetemia, suggesting these subsets may be important in resolving relapsing episodes. In contrast, an early and significant increase in peritoneal B1a, B1b, and B1c cells, but not B2 cells, occurred in response to B. burgdorferi. Later in the course of infection, both species of Borrelia induced the selective expansion of peritoneal B1b cells, suggesting that B1b cells may participate in long-lasting immunity to Lyme and relapsing fever spirochetes. Our data demonstrate that different Borrelia can activate the same B cell subsets in distinct ways and they each elicit a complex interplay of MZ and multiple peritoneal B cell subsets in the early response to infection.
The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation
Authors: Kumar Amrita , Wu Huixia , Collier-Hyams Lauren S. , Kwon Young-Man , Hanson Jason M. , Neish Andrew S.
Published: April 2014

The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-κB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3- SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.
Activated Human Neonatal CD8+ T Cells Are Subject to Immunomodulation by Direct TLR2 or TLR5 Stimulation
Authors: McCarron Mark , Reen Denis J.
Published: April 2014

In conditions of optimal priming, the neonate possess competency to mount quantitatively adult-like responses. Vaccine formulations containing sufficiently potent adjuvants may overcome the neonates’ natural tendency for immunosuppression and provoke a similarly robust immune response. TLR expression on T cells represents the possibility of directly enhancing T cell immunity. We examined the ex vivo responsiveness of highly purified human cord blood-derived CD8+ T cells to direct TLR ligation by a repertoire of TLR agonists. In concert with TCR stimulation, only Pam3Cys (palmitoyl-3-Cys-Ser-(Lys)4) and flagellin monomers significantly enhanced proliferation, CD25+ expression, IL-2, IFN-γ, TNF-α, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8+ T cells. Blocking studies confirmed that the increase in IFN-γ production was by the direct triggering of surface TLR2 or TLR5. The simultaneous exposure of CD8+ T cells to both TLR agonists had an additive effect on IFN-γ production. These data suggest that a combination of the two TLR ligands would be a potent T cell adjuvant. This may represent a new approach to TLR agonist-based adjuvant design for future human neonatal vaccination strategies requiring a CD8+ component.
Immune Complex/Ig Negatively Regulate TLR4-Triggered Inflammatory Response in Macrophages through FcγRIIb-Dependent PGE2 Production
Authors: Zhang Yan , Liu Shuxun , Liu Juan , Zhang Ting , Shen Qian , Yu Yizhi , Cao Xuetao
Published: April 2014

Excessive activation of TLR may induce endotoxin shock and inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Nonpathogenic immune complex (IC) and Ig (IC/Ig) have been shown to play important roles in the regulation of immune responses and to be therapeutic in some kinds of autoimmune diseases. However, the role of IC/Ig in the regulation of TLR-triggered inflammatory responses and the underlying mechanisms remain to be fully understood. In this study we demonstrate that IC/Ig can significantly inhibit LPS-induced secretion of TNF-α and IL-6 from macrophages by preferentially inducing PGE2. Pretreatment of mice with IC can protect wild-type mice, but not FcγRIIb−/− mice, from lethal endotoxin shock, and significantly reduce the levels of serum TNF-α and IL-6 in wild-type mice but not in FcγR IIb−/− mice. Furthermore, blockade of PGE2 by celecoxib restores LPS-induced production of TNF-α and IL-6 in the presence of IC both in vitro and in vivo. Accordingly, blockade of PGE2 production in vivo results in the increased sensitivity of IC-pretreated mice to lethal endotoxin shock. Therefore, IC/Ig can negatively regulate TLR4-triggered inflammatory response in macrophages through FcγRIIb-dependent PGE2. In addition, our results suggest that down-regualtion of NF-κB activation and TLR4 expression but activation of protein kinase A pathway in macrophages by IC/Ig contribute to the negative regulatory process. Thus we provide new manner for the immune regulation and mechanistic explanation for nonpathogenic IC/Ig in the treatment of inflammatory or autoimmune diseases.
Crosstalk between CXCR4/Stromal Derived Factor-1 and VLA-4/VCAM-1 Pathways Regulates Neutrophil Retention in the Bone Marrow
Authors: Petty Joseph M. , Lenox Christopher C. , Weiss Daniel J. , Poynter Matthew E. , Suratt Benjamin T.
Published: April 2014

Neutrophil retention in and release from the bone marrow is a critical process that remains incompletely understood. Previous work has implicated the CXCR4/stromal derived factor-1 (SDF-1) chemokine axis in the marrow retention of neutrophils, yet the adhesion pathways responsible for this retention are unknown. Because α4β1 integrin (VLA-4) and its ligand VCAM-1 play a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether this integrin might be involved in marrow neutrophil retention and release. In this study, we show that VLA-4 is expressed on murine marrow neutrophils and decreases with maturation, whereas blockade of this integrin leads to the release of marrow neutrophils. Marrow neutrophils adhere via VLA-4 to VCAM-1, which is expressed on marrow endothelium and stroma, and inhibition of VCAM-1 causes release of marrow neutrophils. Furthermore, SDF-1 (CXCL12) signaling through neutrophil CXCR4 augments VLA-4 adhesion to VCAM-1 in vitro, an effect that is blocked by preincubation with pertussis toxin. In vivo blockade of both CXCR4 and α4 causes synergistic release of marrow neutrophils, showing that cross-talk between CXCR4 and VLA-4 modulates marrow retention of these cells. Taken together, these results indicate that the VLA-4/VCAM adhesion pathway is critical in the retention and maturation-controlled release of neutrophils from the marrow, while providing an important link between the CXCR4/SDF-1 signaling axis and the adhesion events that govern this process.
Therapeutic Targeting of IL-6 Trans Signaling Counteracts STAT3 Control of Experimental Inflammatory Arthritis
Authors: Nowell Mari A. , Williams Anwen S. , Carty Sarah A. , Scheller Jürgen , Hayes Anthony J. , Jones Gareth W. , Richards Peter J. , Slinn Simon , Ernst Matthias , Jenkins Brendan J. , Topley Nicholas , Rose-John Stefan , Jones Simon A.
Published: April 2014

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.
Reciprocal Expression and Signaling of TLR4 and TLR9 in the Pathogenesis and Treatment of Necrotizing Enterocolitis
Authors: Gribar Steven C. , Sodhi Chhinder P. , Richardson Ward M. , Anand Rahul J. , Gittes George K. , Branca Maria F. , Jakub Adam , Shi Xia-hua , Shah Sohail , Ozolek John A. , Hackam David J.
Published: April 2014

Necrotizing enterocolitis (NEC) is a common and often fatal inflammatory disorder affecting preterm infants that develops upon interaction of indigenous bacteria with the premature intestine. We now demonstrate that the developing mouse intestine shows reciprocal patterns of expression of TLR4 and TLR9, the receptor for bacterial DNA (CpG-DNA). Using a novel ultrasound-guided in utero injection system, we administered LPS directly into the stomachs of early and late gestation fetuses to induce TLR4 signaling and demonstrated that TLR4-mediated signaling within the developing intestine follows its expression pattern. Murine and human NEC were associated with increased intestinal TLR4 and decreased TLR9 expression, suggesting that reciprocal TLR4 and TLR9 signaling may occur in the pathogenesis of NEC. Enteral administration of adenovirus expressing mutant TLR4 to neonatal mice reduced the severity of NEC and increased TLR9 expression within the intestine. Activation of TLR9 with CpG-DNA inhibited LPS-mediated TLR4 signaling in enterocytes in a mechanism dependent upon the inhibitory molecule IRAK-M. Strikingly, TLR9 activation with CpG-DNA significantly reduced NEC severity, whereas TLR9-deficient mice exhibited increased NEC severity. Thus, the reciprocal nature of TLR4 and TLR9 signaling within the neonatal intestine plays a role in the development of NEC and provides novel therapeutic approaches to this disease.
Mast Cells Contribute to Autoimmune Inflammatory Arthritis via Their Tryptase/Heparin Complexes
Authors: Shin Kichul , Nigrovic Peter A. , Crish James , Boilard Eric , McNeil H. Patrick , Larabee Katherine S. , Adachi Roberto , Gurish Michael F. , Gobezie Reuben , Stevens Richard L. , Lee David M.
Published: April 2014

Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase β (hTryptase-β) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-β/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.
Citrullination of CXCL12 Differentially Reduces CXCR4 and CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1 Activity via CXCR4
Authors: Struyf Sofie , Noppen Samuel , Loos Tamara , Mortier Anneleen , Gouwy Mieke , Verbeke Hannelien , Huskens Dana , Luangsay Souphalone , Parmentier Marc , Geboes Karel , Schols Dominique , Van Damme Jo , Proost Paul
Published: April 2014

Posttranslational proteolytic processing of chemokines is a natural mechanism to regulate inflammation. In this study, we describe modification of the CXC chemokine stromal cell-derived factor 1α/CXCL12 by peptidylarginine deiminase (PAD) that converts arginine residues into citrulline (Cit), thereby reducing the number of positive charges. The three NH2-terminal arginines of CXCL12, Arg8, Arg12, and Arg20, were citrullinated upon incubation with PAD. The physiologic relevance of citrullination was demonstrated by showing coexpression of CXCL12 and PAD in Crohn’s disease. Three CXCL12 isoforms were synthesized for biologic characterization: CXCL12-1Cit, CXCL12-3Cit, and CXCL12-5Cit, in which Arg8, Arg8/Arg12/Arg20, or all five arginines were citrullinated, respectively. Replacement of only Arg8 caused already impaired (30-fold reduction) CXCR4 binding and signaling (calcium mobilization, phosphorylation of ERK and protein kinase B) properties. Interaction with CXCR4 was completely abolished for CXCL12-3Cit and CXCL12-5Cit. However, the CXCR7-binding capacities of CXCL12-1Cit and CXCL12-3Cit were, respectively, intact and reduced, whereas CXCL12-5Cit failed to bind CXCR7. In chemotaxis assays with lymphocytes and monocytes, CXCL12-3Cit and CXCL12-5Cit were completely devoid of activity, whereas CXCL12-1Cit, albeit at higher concentrations than CXCL12, induced migration. The antiviral potency of CXCL12-1Cit was reduced compared with CXCL12 and CXCL12-3Cit and CXCL12-5Cit (maximal dose 200 nM) could not inhibit infection of lymphocytic MT-4 cells with the HIV-1 strains NL4.3 and HE. In conclusion, modification of CXCL12 by one Cit severely impaired the CXCR4-mediated biologic effects of this chemokine and maximally citrullinated CXCL12 was inactive. Therefore, PAD is a potent physiologic down-regulator of CXCL12 function.
Heat Shock Proteins HSP27 and HSP70 Are Present in the Skin and Are Important Mediators of Allergic Contact Hypersensitivity
Authors: Yusuf Nabiha , Nasti Tahseen H. , Huang Chun-Ming , Huber Brad S. , Jaleel Tarannum , Lin Hui-Yi , Xu Hui , Elmets Craig A.
Published: April 2014

Proteomic analysis of murine skin has shown that a variety of heat shock proteins (HSPs) are constitutively expressed in the skin. Using murine allergic contact hypersensitivity as a model, we investigated the role of two heat shock proteins, HSP27 and HSP70, in the induction of cutaneous cell-mediated immune responses. Immunohistochemical examination of skin specimens showed that HSP27 was present in the epidermis and HSP70 was present in both the epidermis and dermis. Inhibition of HSP27 and HSP70 produced a reduction in the 1-fluoro-2,4-dinitrobenzene contact hypersensitivity response and resulted in the induction of Ag-specific unresponsiveness. Treatment of dendritic cell cultures with recombinant HSP27 caused in the up-regulation of IL-1β, TNF-α, IL-6, IL-12p70, and IL-12p40 but not IL-23p19, which was inhibited when Abs to HSP27 were added. The 1-fluoro-2,4-dinitrobemzene-conjugated dendritic cells that had been treated with HSP27 had an increased capacity to initiate contact hypersensitivity responses compared with control dendritic cells. This augmented capacity required TLR4 signaling because neither cytokine production by dendritic cells nor the increased induction of contact hypersensitivity responses occurred in TLR4-deficient C3H/HeJ mice. Our findings indicate that a cascade of events occurs following initial interaction of hapten with the skin that includes increased activity of HSPs, their interaction with TLR4, and, in turn, increased production of cytokines that are known to enhance Ag presentation by T cells. The results suggest that HSPs form a link between adaptive and innate immunity during the early stages of contact hypersensitivity.
Targeting TLR2 Attenuates Pulmonary Inflammation and Fibrosis by Reversion of Suppressive Immune Microenvironment
Authors: Yang Hong-Zhen , Cui Bing , Liu Han-Zhi , Chen Zhi-Rong , Yan Hui-Min , Hua Fang , Hu Zhuo-Wei
Published: April 2014

Pulmonary fibrosis is a consequence of chronic lung injury and is associated with a high mortality. Despite the pathogenesis of pulmonary fibrosis remaining as an enigma, immune responses play a critical role in the deregulation of wound healing process after lung injury, which leads to fibrosis. Accumulating evidence argues the rationales for current treatments of pulmonary fibrosis using immunosuppressive agents such as corticosteroids. In this study, we report that bleomycin (BLM), a well-known fibrogenic agent functioning as a TLR2 agonist, induced the maturation of dendritic cells and release of cytokines. The BLM activation of TLR2 mediated a time-dependent alteration of immune responses in the lung. These responses resulted in an increase in the tissue-infiltrating proinflammatory cells and cytokines in the early period initially following BLM exposure and an increase in the tissue-infiltrating suppressive immune cells and factors during the later period following BLM exposure. TLR2 deficiency, however, reduced pulmonary inflammation, injury, and subsequently attenuated pulmonary fibrosis. Targeting TLR2 by a TLR2-neutralizing Ab not only markedly decreased animal death but also protected animals from the development of pulmonary fibrosis and reversed the established pulmonary fibrosis through regulating BLM-induced immunosuppressive microenvironments. Our studies suggest that TLR2 is a promising target for the development of therapeutic agents against pulmonary fibrosis and that eliminating immunosuppressive cells and factors via immunostimulants is a novel strategy for fibro-proliferative diseases. Moreover, combining BLM with an anti-TLR2 Ab or TLR2 antagonist for cancer therapy will improve the BLM therapeutic profile by enhancing anti-cancer efficacy and reducing systemic inflammation and pulmonary fibrosis.
Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia Stimulate Peripheral Blood Mononuclear Cells to Secrete TNF-α and IFN-γ
Authors: Takamatsu Hiroyuki , Espinoza J. Luis , Lu Xuzhang , Qi Zhirong , Okawa Katsuya , Nakao Shinji
Published: April 2014

Moesin is an intracellular protein that links the cell membrane and cytoskeleton, while also mediating the formation of microtubules and cell adhesion sites as well as ruffling of the cell membrane. To determine the roles of anti-moesin Abs derived from the serum of patients with aplastic anemia (AA) in the pathophysiology of bone marrow failure, we studied the expression of moesin on various blood cells and the effects of anti-moesin Abs on the moesin-expressing cells. The proteins recognized by anti-moesin mAbs were detectable on the surface of T cells, NK cells, and monocytes from healthy individuals as well as on THP-1 cells. The peptide mass fingerprinting of the THP-1 cell surface protein and the knock-down experiments using short hairpin RNA proved that the protein is moesin itself. Both the anti-moesin mAbs and the anti-moesin polyclonal Abs purified from the AA patients’ sera stimulated THP-1 cells and the PBMCs of healthy individuals and AA patients to secrete 60–80% as much TNF-α as did LPS 100 ng/ml. Although the polyclonal Abs induced IFN-γ secretion from the PBMCs of healthy individuals only when the PBMCs were prestimulated by anti-CD3 mAbs, the anti-moesin Abs were capable of inducing IFN-γ secretion from the PBMCs of AA patients by themselves. Anti-moesin Abs may therefore indirectly contribute to the suppression of hematopoiesis in AA patients by inducing myelosuppressive cytokines from immunocompetent cells.
Airway Epithelial Cells Regulate the Functional Phenotype of Locally Differentiating Dendritic Cells: Implications for the Pathogenesis of Infectious and Allergic Airway Disease
Authors: Rate Angela , Upham John W. , Bosco Anthony , McKenna Kathy L. , Holt Patrick G.
Published: April 2014

Atopic asthma pathogenesis is driven by the combined effects of airway inflammation generated during responses to viral infections and aeroallergens, and both these pathways are regulated by dendritic cells (DC) that differentiate locally from monocytic precursors. These DCs normally exhibit a sentinel phenotype characterized by active Ag sampling but attenuated presentation capability, which limits the intensity of local expression of adaptive immunity. How this tight control of airway DC functions is normally maintained, and why it breaks down in some atopics leading to immunopathological changes in airway tissues, is unknown. We postulated that signals from adjacent airway epithelial cells (AEC) contribute to regulation of local differentiation of DC. We tested this in a coculture model containing both cell types in a GM-CSF-IL-4-enriched cytokine milieu characteristic of the atopic asthmatic airway mucosa. We demonstrate that contact with AEC during DC differentiation up-regulates expression of the function-associated markers MHC class II, CD40, CD80, TLR3, and TLR4 on DCs with concomitant up-regulation of Ag uptake/processing. Moreover, the AEC-conditioned DCs displayed increased LPS responsiveness evidenced by higher production of IL-12, IL-6, IL-10, and TNF-α. The Th2 memory-activating properties of AEC-conditioned DCs were also selectively attenuated. Data from microarray and blocking experiments implicate AEC-derived type 1 IFNs and IL-6 in modulation of DC differentiation. Collectively, these findings suggest that resting AECs modulate local DC differentiation to optimize antimicrobial defenses in the airways and in the process down-modulate capacity for expression of potentially damaging Th2 immunity.
App1: An Antiphagocytic Protein That Binds to Complement Receptors 3 and 2
Authors: Stano Paola , Williams Virginia , Villani Maristella , Cymbalyuk Eugene S. , Qureshi Asfia , Huang Yuxiang , Morace Giulia , Luberto Chiara , Tomlinson Stephen , Del Poeta Maurizio
Published: April 2014

In previous studies, we showed that the pathogenic fungus Cryptococcus neoformans (Cn) produces a specific and unique protein called antiphagocytic protein 1 (App1), which inhibits phagocytosis of Cn by alveolar macrophages (AMs). Phagocytosis of Cn by AMs occurs mainly through a complement- or Ab-mediated mechanism. Among AM receptors, complement receptor 3 (CR3) and FcRγ are the most common receptors involved in the phagocytic process. Because App1 inhibits phagocytosis of complement- but not Ab-coated erythrocytes, we investigated the role of CR3 in App1-macrophage interactions. We found that App1 binds to CR3 and if CR3 is absent from the surface of AMs, its antiphagocytic action is lost. When we investigated whether App1 would also bind to other complement receptor(s), we found that App1 does bind to complement receptor 2 (CR2) in a dose-dependent manner. In certain lymphoma cell lines, cellular proliferation is stimulated by complement through CR2, providing a potential use of App1 as a proliferation inhibitor of these cells. Initially discovered as an antiphagocytic protein regulating CR3-mediated innate immunity, App1 may also play a key role in the regulation of acquired immunity, because CR2 is mainly localized on B cells.
Ezrin and Moesin Function Together to Promote T Cell Activation
Authors: Shaffer Meredith H. , Dupree Renell S. , Zhu Peimin , Saotome Ichiko , Schmidt Richard F. , McClatchey Andrea I. , Freedman Bruce D. , Burkhardt Janis K.
Published: April 2014

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin−/− T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-θ, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin−/− T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-γ1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.
14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation
Authors: Ajjappala Brijesh S. , Kim Yong-Soo , Kim Myung-Sun , Lee Min-Young , Lee Kwang-Youl , Ki Ho-Yun , Cha Dong-Hyun , Baek Kwang-Hyun
Published: April 2014

IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3γ was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3γ overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3γ in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3γ, resulting in cell death. These results indicate that deregulated expression of 14-3-3γ may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.
Cell-Mediated Immunity toToxoplasma gondiiDevelops Primarily by Local Th1 Host Immune Responses in the Absence of Parasite Replication
Authors: Gigley Jason P. , Fox Barbara A. , Bzik David J.
Published: April 2014

A single inoculation of mice with the live, attenuated Toxoplasma gondii uracil auxotroph strain cps1-1 induces long-lasting immunity against lethal challenge with hypervirulent strain RH. The mechanism for this robust immunity in the absence of parasite replication has not been addressed. The mechanism of long-lasting immunity, the importance of route of immunization, cellular recruitment to the site of infection, and local and systemic inflammation were evaluated. Our results show that infection with cps1-1 elicits long-lasting CD8+ T cell- mediated immunity. We show that immunization with cps1-1-infected dendritic cells elicits long-lasting immunity. Intraperitoneal infection with cps1-1 induced a rapid influx of GR1+ neutrophils and two stages of GR1+CD68+ inflammatory monocyte infiltration into the site of inoculation. CD19+ B cells and CD3+ T cells steadily increase for 8 days after infection. CD8+ T cells were rapidly recruited to the site of infection and increased faster than CD4+ T cells. Surprisingly, cps1-1 infection induced high systemic levels of bioactive IL-12p70 and a very low level and transient systemic IFN-γ. Furthermore, we show significant levels of these inflammatory cytokines were locally produced at the site of cps1-1 inoculation. These findings offer new insight into immunological mechanisms and local host responses to a non-replicating type I parasite infection associated with development of long-lasting immunity to Toxoplasma gondii.
Induction of Proinflammatory Cytokines in Primary Human Macrophages by Influenza A Virus (H5N1) Is Selectively Regulated by IFN Regulatory Factor 3 and p38 MAPK
Authors: Hui Kenrie P. Y. , Lee Suki M. Y. , Cheung Chung-yan , Ng Iris H. Y. , Poon Leo L. M. , Guan Yi , Ip Nancy Y. Y. , Lau Allan S. Y. , Peiris J. S. Malik
Published: April 2014

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-α, IFN-β, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-α, IFN-β, and IFN-λ1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-β, IFN-λ1, and MCP-1 but only to a partial reduction of TNF-α. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-β, IFN-λ1, and MCP-1 but only partly control TNF-α induction. A more precise identification of the differences in the regulation of TNF-α and IFN-β could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.
Type I IFN-Mediated Protection of Macrophages and Dendritic Cells Secures Control of Murine Coronavirus Infection
Authors: Cervantes-Barragán Luisa , Kalinke Ulrich , Züst Roland , König Martin , Reizis Boris , López-Macías Constantino , Thiel Volker , Ludewig Burkhard
Published: April 2014

The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses. Plasmacytoid dendritic cell-derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus (MHV). The aim of this study was to determine the major target cell populations of this first wave of type I IFNs. Generation of bone marrow-chimeric mice expressing the type I IFN receptor (IFNAR) on either hemopoietic or non-bone marrow-derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells. To establish which cell population responds most efficiently to type I IFNs, mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV. This genetic analysis revealed that IFNAR expression on LysM+ macrophages and CD11c+ dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease. This study identifies type I IFN-mediated cross-talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other, as an essential cellular pathway for the control of fatal cytopathic virus infection.
Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells
Authors: Barrett Nora A. , Maekawa Akiko , Rahman Opu M. , Austen K. Frank , Kanaoka Yoshihide
Published: April 2014

House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.
P2Y6 Receptors Require an Intact Cysteinyl Leukotriene Synthetic and Signaling System to Induce Survival and Activation of Mast Cells
Authors: Jiang Yongfeng , Borrelli Laura , Bacskai Brian J. , Kanaoka Yoshihide , Boyce Joshua A.
Published: April 2014

Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors and activate mast cells in vitro. We previously demonstrated that cys-LTs cross-desensitized IL-4-primed primary human mast cells (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. Leukotriene (LT) D4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of ERK and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase, implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of MIP-1β, a response blocked by short hairpin RNA-mediated knockdown of CysLT1R or P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are coexpressed on many cell types of innate immunity, reciprocally amplify one another’s function in mast cells through endogenous ligands.
Increased Reactivity of Dendritic Cells from Aged Subjects to Self-Antigen, the Human DNA
Authors: Agrawal Anshu , Tay Jia , Ton Steven , Agrawal Sudhanshu , Gupta Sudhir
Published: April 2014

Diminished immune functions and chronic inflammation are hallmarks of aging. The underlying causes are not well understood. In this investigation, we show an increased reactivity of dendritic cells (DCs) from aged subjects to self-Ags as one of the potential mechanisms contributing to age-associated inflammation. Consistent with this, DCs from aged subjects display increased reactivity to intracellular human DNA, a self-Ag, by secreting enhanced quantities of type I IFN and IL-6 compared with the DCs from young subjects. Furthermore, this is accompanied by an increased up-regulation of costimulatory molecules CD80 and CD86. These DNA-primed DCs from aged subjects enhanced T cell proliferation compared with the young subjects, further substantiating our findings. Investigations of signaling mechanisms revealed that DNA-stimulated DCs from aged subjects displayed a significantly higher level of IFN regulatory factor-3 and NF-κB activity compared with their young counterparts. More importantly, DCs from aged subjects displayed a higher level of NF-κB activation at the basal level, suggesting an increased state of activation. This activated state of DCs may be responsible for their increased reactivity to self-Ags such as DNA, which in turn contributes to the age-associated chronic inflammation.