bluuebunny/crossref_metadata_2025 huggingface dataset:
An Essential Role of Sialylated O-Linked Sugar Chains in the Recognition of Mouse CD99 by Paired Ig-Like Type 2 Receptor (PILR)
Authors: Wang Jing , Shiratori Ikuo , Satoh Takeshi , Lanier Lewis L. , Arase Hisash
Published: April 2014

The paired Ig-like type 2 receptor (PILR), which comprises both inhibitory and activating isoforms, is well conserved among most mammalians. The inhibitory PILRα possesses an ITIM in its cytoplasmic domain, whereas the activating PILRβ does not have an ITIM but transduces activating signals by associating with the ITAM-bearing DAP12 adapter molecule. Both mouse PILRα and PILRβ recognize mouse CD99, which is broadly expressed on various cells, including lymphocytes, and is involved in the regulation of immune responses. We herein report that sialylated O-linked sugar chains on CD99 are essential for the recognition by PILR. Mutations of one of two O-glycosylation sites on CD99 significantly reduced recognition of CD99 by the activating PILRβ, whereas recognition by the inhibitory PILRα was not affected. In contrast, mutations of both O-glycosylation sites on CD99 completely abrogated the recognition by both PILRα and PILRβ. PILR did not recognize CD99 treated with neuraminidase, and CD99 expressed on cells transfected with core 2 β-1,6-N-acetylglucosaminyltransferase was not recognized by PILR. NK cells expressing endogenous activating PILRβ receptors mediated cytotoxicity against cells expressing wild-type CD99 but not cells expressing mutant CD99 that lacked O-glycosylation sites. These findings indicate that sialylated O-linked sugar structures on CD99 play an important role in the recognition of PILR.
Puromycin-Sensitive Aminopeptidase Limits MHC Class I Presentation in Dendritic Cells but Does Not Affect CD8 T Cell Responses during Viral Infections
Authors: Towne Charles F. , York Ian A. , Neijssen Joost , Karow Margaret L. , Murphy Andrew J. , Valenzuela David M. , Yancopoulos George D. , Neefjes Jacques J. , Rock Kenneth L.
Published: April 2014

Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice. In addition, dendritic cells from PSA-deficient mice display more MHC class I molecules on the cell surface, suggesting that PSA normally limits Ag presentation by destroying certain peptides in these key APCs. Surprisingly, MHC class I levels are not altered on other PSA-deficient cells and the processing and presentation of peptide precursors in PSA-deficient fibroblasts is normal. Moreover, PSA-deficient mice have normal numbers of T cells in the periphery, and respond as well as wild type mice to eight epitopes from three viruses. These data indicate that PSA may play a role in limiting MHC class I Ag presentation in dendritic cells in vivo but that it is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags.
Identification of Pax5 Target Genes in Early B Cell Differentiation
Authors: Pridans Clare , Holmes Melissa L. , Polli Matthew , Wettenhall James M. , Dakic Aleksandar , Corcoran Lynn M. , Smyth Gordon K. , Nutt Stephen L.
Published: April 2014

The transcription factor Pax5 is essential for B cell commitment in the mouse, where it represses lineage-inappropriate gene expression while simultaneously activating the B cell gene expression program. In this study we have performed a global gene expression screen of wild-type and Pax5-deficient pro-B cells in an attempt to identify the crucial Pax5 targets in early B lymphopoiesis. These studies have identified 109 Pax5 targets comprising 61% activated and 39% repressed genes. Interestingly, Pax5 directly regulates the genes encoding a number of transcription factors that are required at the pre-B cell stage of differentiation, including Irf8, Spib, and Ikzf3 (Aiolos), suggesting that a key function of Pax5 is to activate secondary transcription factors that further reinforce the B cell program. Pax5 is also required for the expression of many genes known to be involved in adhesion and signaling, indicating that Pax5 modulates the homing and or migration properties of B cell progenitors. Finally, Pax5 also represses a cohort of genes that are involved in multiple biological processes, many of which are not typically associated with B cells. These include the repression of the adhesion molecule Embigin, which is expressed in bone marrow progenitors, T cells, and myeloid cells but is specifically repressed by Pax5 in B cells.
Healthy Human Subjects Have CD4+ T Cells Directed against H5N1 Influenza Virus
Authors: Roti Michelle , Yang Junbao , Berger DeAnna , Huston Laurie , James Eddie A. , Kwok William W.
Published: April 2014

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4+ T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4+ T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4+ T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-γ, TNF-α, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.
Naturally Occurring IgM Anti-Leukocyte Autoantibodies (IgM-ALA) Inhibit T Cell Activation and Chemotaxis
Authors: Lobo Peter I. , Schlegel Kailo H. , Spencer Clinton E. , Okusa Mark D. , Chisholm Christopher , Mchedlishvili Nino , Park Andrew , Christ Constance , Burtner Christopher
Published: April 2014

The physiological relevance of naturally occurring IgM-ALA remains to be elucidated. These autoantibodies are present from birth and increase in diverse inflammatory states that are both infectious and noninfectious. Clinical observations showing significantly less acute allograft rejections in recipients having high IgM-ALA levels, led us to investigate whether IgM-ALA could have a functional role in attenuating T cell mediated inflammatory responses. In pursuit of this hypothesis, we did studies using IgM purified from the serum of normal individuals, patients with end stage renal disease, and HIV-1 infection. All preparations of IgM immunoprecipitated certain receptors e.g., CD3, CD4, CCR5, and CXCR4 from whole cell lysates but failed to immunoprecipitate IL-2R and HLA Ags. In physiological doses IgM down-regulated CD4, CD2 and CD86 but not CD8 and CD28, inhibited T cell proliferation, decreased production of certain proinflammatory cytokines e.g., TNF-α, IL-13 and IL-2, but not IFN- γ, IL-1β, GM-CSF, IL-6 and IL-8 and inhibited leukocyte chemotaxis. These inhibitory effects were more pronounced when using IgM from patients with high levels of IgM-ALA and these inhibitory effects were significantly reduced after using IgM preabsorbed with leukocytes. IgM-ALA binding to leukocytes was found to be highly specific, as <10% of IgM secreting B cell clones had IgM-ALA specificity with some clones having specificity for either T cells or monocytes. These findings support the concept that IgM-ALA provides an innate mechanism to regulate T cell mediated inflammatory responses.
Down-Regulation of MHC II in Mesenchymal Stem Cells at High IFN-γ Can Be Partly Explained by Cytoplasmic Retention of CIITA
Authors: Tang Katherine C. , Trzaska Katarzyna A. , Smirnov Sergey V. , Kotenko Sergei V. , Schwander Stephan K. , Ellner Jerrold J. , Rameshwar Pranela
Published: April 2014

Mesenchymal stem cells (MSCs) are located in postnatal bone marrow, show plasticity, are linked to various bone marrow disorders, exhibit phagocytosis, exert Ag-presenting properties (APC), and are immune suppressive. Unlike professional APCs, MSCs respond bimodally to IFN-γ in MHC-II expression, with expression at 10 U/ml and baseline, and down-regulation at 100 U/ml. The effects at high IFN-γ could not be explained by down-regulation of its receptor, IFN-γRI. In this study, we report on the mechanisms by which IFN-γ regulates MHC-II expression in MSCs. Gel shift assay and Western blot analyses showed dose-dependent increases in activated STAT-1, indicating responsiveness by IFN-γRI. Western blots showed decreased intracellular MHC-II, which could not be explained by decreased transcription of the master regulator CIITA, based on RT-PCR and in situ immunofluorescence. Reporter gene assays with PIII and PIV CIITA promoters indicate constitutive expression of PIII in MSCs and a switch to PIV by IFN-γ, indicating the presence of factors for effect promoter responses. We explained decreased MHC-II at the level of transcription because CIITA protein was observed in the cytosol and not in nuclei at high IFN-γ level. The proline/serine/threonine region of CIITA showed significant decrease in phosphorylation at high IFN-γ levels. An understanding of the bimodal effects could provide insights on bone marrow homeostasis, which could be extrapolated to MSC dysfunction in hematological disorders.
The Myeloid Differentiation Factor 88 (MyD88) Is Required for CD4+ T Cell Effector Function in a Murine Model of Inflammatory Bowel Disease
Authors: Fukata Masayuki , Breglio Keith , Chen Anli , Vamadevan Arunan S. , Goo Tyralee , Hsu David , Conduah Daisy , Xu Ruliang , Abreu Maria T.
Published: April 2014

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4+CD45Rbhigh T cells and/or CD4+CD45RblowCD25+ regulatory T cells were isolated and adoptively transferred to RAG1−/− mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [3H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4+CD45Rbhigh T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88−/− CD4+ T cells showed decreased proliferation compared with WT CD4+ T cells both in vivo and in vitro. CD4+CD45Rbhigh T cells from MyD88−/− mice did not induce wasting disease when transferred into RAG1−/− recipients. Lamina propria CD4+ T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88−/− cells than mice receiving WT cells. In vitro, MyD88−/− T cells were blunted in their ability to secrete IL-17 but not IFN-γ. Absence of MyD88 in CD4+CD45Rbhigh cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.
Dual Roles of Immunoregulatory Cytokine TGF-β in the Pathogenesis of Autoimmunity-Mediated Organ Damage
Authors: Saxena Vijay , Lienesch Douglas W. , Zhou Min , Bommireddy Ramireddy , Azhar Mohamad , Doetschman Thomas , Singh Ram Raj
Published: April 2014

Ample evidence suggests a role of TGF-β in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-β1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F1 mice have reduced expression of TGF-β1 in lymphoid tissues, and TGF-β1 or TGF-β1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-β1 protein and mRNA and TGF-β signaling proteins (TGF-β receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-β1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-β blockade by treatment of these mice with an anti-TGF-β Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-β plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-β in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-β in target organs to counter inflammation. Enhanced TGF-β in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.
Differential Contribution of Three Activating IgG Fc Receptors (FcγRI, FcγRIII, and FcγRIV) to IgG2a- and IgG2b-Induced Autoimmune Hemolytic Anemia in Mice
Authors: Baudino Lucie , Nimmerjahn Falk , Azeredo da Silveira Samareh , Martinez-Soria Eduardo , Saito Takashi , Carroll Michael , Ravetch Jeffrey V. , Verbeek J. Sjef , Izui Shozo
Published: April 2014

Murine phagocytes express three different activating IgG FcγR: FcγRI is specific for IgG2a; FcγRIII for IgG1, IgG2a, and IgG2b; and FcγRIV for IgG2a and IgG2b. Although the role of FcγRIII in IgG1 and IgG2a anti-RBC-induced autoimmune hemolytic anemia (AIHA) is well documented, the contribution of FcγRI and FcγRIV to the development of IgG2a- and IgG2b-induced anemia has not yet been defined. In the present study, using mice deficient in FcγRI, FcγRIII, and C3, in combination with an FcγRIV-blocking mAb, we assessed the respective roles of these three FcγR in the development of mild and severe AIHA induced by two different doses (50 and 200 μg) of the IgG2a and IgG2b subclasses of the 34-3C anti-RBC monoclonal autoantibody. We observed that the development of mild anemia induced by a low dose of 34-3C IgG2a autoantibody was highly dependent on FcγRIII, while FcγRI and FcγRIV additionally contributed to the development of severe anemia induced by a high dose of this subclass. In contrast, the development of both mild and severe anemia induced by 34-3C IgG2b was dependent on FcγRIII and FcγRIV. Our results indicate differential roles of the three activating FcγR in IgG2a- and IgG2b-mediated AIHA.
Cutting Edge: A cis-Acting DNA Element Targets AID-Mediated Sequence Diversification to the Chicken Ig Light Chain Gene Locus
Authors: Kothapalli Nagarama , Norton Darrell D. , Fugmann Sebastian D.
Published: April 2014

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3′ regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.
Cutting Edge: Innate Immune Response Triggered by Influenza A Virus Is Negatively Regulated by SOCS1 and SOCS3 through a RIG-I/IFNAR1-Dependent Pathway
Authors: Pothlichet Julien , Chignard Michel , Si-Tahar Mustapha
Published: April 2014

Influenza A virus (IAV) triggers a contagious respiratory disease that produces considerable lethality. Although this lethality is likely due to an excessive host inflammatory response, the negative feedback mechanisms aimed at regulating such a response are unknown. In this study, we investigated the role of the eight “suppressor of cytokine signaling” (SOCS) regulatory proteins in IAV-triggered cytokine expression in human respiratory epithelial cells. SOCS1 to SOCS7, but not cytokine-inducible Src homology 2-containing protein (CIS), are constitutively expressed in these cells and only SOCS1 and SOCS3 expressions are up-regulated upon IAV challenge. Using distinct approaches affecting the expression and/or the function of the IFNαβ receptor (IFNAR)1, the viral sensors TLR3 and retinoic acid-inducible gene I (RIG-I) as well as the mitochondrial antiviral signaling protein (MAVS, a RIG-I signaling intermediate), we demonstrated that SOCS1 and SOCS3 up-regulation requires a TLR3-independent, RIG-I/MAVS/IFNAR1-dependent pathway. Importantly, by using vectors overexpressing SOCS1 and SOCS3 we revealed that while both molecules inhibit antiviral responses, they differentially modulate inflammatory signaling pathways.
Critical Roles of the p110β Subtype of Phosphoinositide 3-Kinase in Lipopolysaccharide-Induced Akt Activation and Negative Regulation of Nitrite Production in RAW 264.7 Cells
Authors: Tsukamoto Ken , Hazeki Kaoru , Hoshi Megumi , Nigorikawa Kiyomi , Inoue Norimitsu , Sasaki Takehiko , Hazeki Osamu
Published: April 2014

It has been suggested that PI3K participates in TLR signaling. However, identifying specific roles for individual PI3K subtypes in signaling has remained elusive. In macrophages from the p110γ−/− mouse, LPS-induced phosphorylation of Akt occurred normally despite the fact that the action of anaphylatoxin C5a was impaired markedly. In RAW 264.7 cells expressing short hairpin RNA that targets p110β, LPS-induced phosphorylation of Akt was significantly attenuated. In contrast, the LPS action was not impaired, but was rather augmented in the p110α-deficient cells. Previous pharmacologic studies have suggested that a PI3K-Akt pathway negatively regulates TLR-induced inducible NO synthase expression and cytokine production. In the p110β-deficient cells, inducible NO synthase expression and IL-12 production upon stimulation by LPS were increased, whereas LPS-induced expression of COX-2 and activation of MAPKs were unaffected. Together, the results suggest a specific function of p110β in the negative feedback regulation of TLR signaling.
pConsensus Peptide Induces Tolerogenic CD8+ T Cells in Lupus-Prone (NZB × NZW)F1 Mice by Differentially Regulating Foxp3 and PD1 Molecules
Authors: Singh Ram Pyare , La Cava Antonio , Hahn Bevra H.
Published: April 2014

Systemic lupus erythematosus is an autoimmune disease caused primarily by autoantibodies (including IgG anti-DNA) and immune complexes that cause tissue damage. After tolerization with an artificial peptide (pConsensus, pCons) based on murine anti-DNA IgG sequences containing MHC class I and class II T cell determinants, lupus-prone (NZB × NZW)F1 female (BWF1) mice develop regulatory CD4+CD25+ T cells and inhibitory CD8+ T cells, both of which suppress anti-DNA Ig production and immune glomerulonephritis. In the present work, we show that splenocytes from BWF1 mice treated with pCons had significant expansion of primarily CD8+ T cells. CD4+ T cells and B cells were each directly suppressed by CD8+ T cells from tolerized mice in a contact-independent manner. Both pCons-induced CD8+CD28+ and CD8+CD28− T cells suppressed production of anti-DNA in vitro. Silencing with small interfering RNA of Foxp3 abrogated the suppression mediated by both CD8+ T cell subsets. Additionally, CD8+ T cells from tolerized mice were weakly cytotoxic against syngeneic B cells from old anti-DNA-producing mice, but not from young mice. Importantly, pCons treatment had dual effects on CD8+ suppressor T cells from tolerized mice, increasing the intracellular expression of Foxp3 while decreasing the surface expression of PD1 molecules. Blocking PD1/PDL1 interactions in the CD8+ T cells from tolerized mice reduced their expression of Foxp3 and their ability to suppress CD4+CD25− proliferation. In contrast, blocking PD1/PDL1 in naive T cells increased Foxp3 expression. Our data suggest that tolerization with pCons activates different subsets of inhibitory/cytotoxic CD8+ T cells whose targets are both CD4+CD25− effector T cells and B cells.
IL-12 Is Required for Anti-OX40-Mediated CD4 T Cell Survival
Authors: Ruby Carl E. , Montler Ryan , Zheng Rongxui , Shu Suyu , Weinberg Andrew D.
Published: April 2014

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rβ2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rβ2 on OX40-stimulated CD4 T cells was tightly regulated and peaked ∼4–6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rβ2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.
Chlamydia muridarum Infection Subverts Dendritic Cell Function to Promote Th2 Immunity and Airways Hyperreactivity
Authors: Kaiko Gerard E. , Phipps Simon , Hickey Danica K. , Lam Chuan En , Hansbro Philip M. , Foster Paul S. , Beagley Kenneth W.
Published: April 2014

There is strong epidemiological evidence that Chlamydia infection can lead to exacerbation of asthma. However, the mechanism(s) whereby chlamydial infection, which normally elicits a strong Th type 1 (Th1) immune response, can exacerbate asthma, a disease characterized by dominant Th type 2 (Th2) immune responses, remains unclear. In the present study, we show that Chlamydia muridarum infection of murine bone marrow-derived dendritic cells (BMDC) modulates the phenotype, cytokine secretion profile, and Ag-presenting capability of these BMDC. Chlamydia-infected BMDC express lower levels of CD80 and increased CD86 compared with noninfected BMDC. When infected with Chlamydia, BMDC secrete increased TNF-α, IL-6, IL-10, IL-12, and IL-13. OVA peptide-pulsed infected BMDC induced significant proliferation of transgenic CD4+ DO11.10 (D10) T cells, strongly inhibited IFN-γ secretion by D10 cells, and promoted a Th2 phenotype. Intratracheal transfer of infected, but not control noninfected, OVA peptide-pulsed BMDC to naive BALB/c mice, which had been i.v. infused with naive D10 T cells, resulted in increased levels of IL-10 and IL-13 in bronchoalveolar lavage fluid. Recipients of these infected BMDC showed significant increases in airways resistance and decreased airways compliance compared with mice that had received noninfected BMDC, indicative of the development of airways hyperreactivity. Collectively, these data suggest that Chlamydia infection of DCs allows the pathogen to deviate the induced immune response from a protective Th1 to a nonprotective Th2 response that could permit ongoing chronic infection. In the setting of allergic airways inflammation, this infection may then contribute to exacerbation of the asthmatic phenotype.
Histone Methylation Patterns Are Cell-Type Specific in Human Monocytes and Lymphocytes and Well Maintained at Core Genes
Authors: Miao Feng , Wu Xiwei , Zhang Lingxiao , Riggs Arthur D. , Natarajan Rama
Published: April 2014

Different immune cells are expected to have unique, obligatory, and stable epigenomes for cell-specific functions. Histone methylation is recognized as a major layer of the cellular epigenome. However, the discovery of histone demethylases raises questions about the stability of histone methylation and its role in the epigenome. In this study, we used chromatin-immunoprecipitation combined with microarrays to map histone H3K9 dimethylation (H3K9Me2) patterns in gene coding and CpG island regions in human primary monocytes and lymphocytes. This chromosomal mark showed consistent distribution patterns in either monocytes or lymphocytes from multiple volunteers despite age or gender, but the pattern in monocytes was clearly distinct from lymphocytes of the same population. Gene Set Enrichment analysis, a bioinformatics tool, revealed that H3K9Me2 candidate genes are enriched in many tightly controlled signaling and cell-type specific pathways. These results demonstrate that monocytes and lymphocytes have distinct epigenomes and H3K9Me2 may play regulatory roles in the transcription of genes indispensable for maintaining immune responses and cell-type specificity.
Productive Coupling of Accessible Vβ14 Segments and DJβ Complexes Determines the Frequency of Vβ14 Rearrangement
Authors: Ranganath Sheila , Carpenter Andrea C. , Gleason Megan , Shaw Albert C. , Bassing Craig H. , Alt Frederick W.
Published: April 2014

To elucidate mechanisms that regulate Vβ rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dβ-Jβ segments inserted into the endogenous Vβ14 locus (Vβ14Rep). As a control, we first generated and analyzed mice with the same Dβ-Jβ cassette targeted into the generally expressed c-myc locus (c-mycRep). Substantial c-mycRep recombination occurred in both T and B cells and initiated concurrently with endogenous Dβ to Jβ rearrangements in thymocytes. In contrast, Vβ14Rep recombination was restricted to T cells and initiated after endogenous Dβ to Jβ rearrangements, but concurrently with endogenous Vβ14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vβ14 rearrangements occur on only 5% of endogenous TCRβ alleles, the Vβ14Rep cassette underwent rearrangement on 80–90% of alleles, supporting the suggestion that productive coupling of accessible Vβ14 segments and DJβ complexes influence the frequency of Vβ14 rearrangements. Strikingly, Vβ14Rep recombination also occurs on TCRβ alleles lacking endogenous Vβ to DJβ rearrangements, indicating that Vβ14 accessibility per se is not subject to allelic exclusion.
Interferon-Induced Expression of MxA in the Respiratory Tract of Rhesus Macaques Is Suppressed by Influenza Virus Replication
Authors: Carroll Timothy D. , Matzinger Shannon R. , Genescà Meritxell , Fritts Linda , Colòn Roxana , McChesney Michael B. , Miller Christopher J.
Published: April 2014

To determine the relationship between influenza A virus replication and innate antiviral immune responses, rhesus monkeys were given oseltamivir before influenza A/Memphis/7/01 (H1N1) challenge. We found that oseltamivir treatment significantly reduced viral replication in the trachea (p < 0.029). Further, in the trachea of both treated and untreated monkeys the mRNA levels of most innate antiviral molecules in the IFN-αβ pathway were dramatically increased by 24 h postinfection. However, the mRNA level of a single IFN-stimulated gene, MxA (myxovirus resistance A), the IFN-stimulated gene known to be critical in blocking influenza virus replication, was significantly lower in the tracheal lavages of untreated monkeys than in the oseltamivir-treated monkeys (p = 0.05). These results demonstrate for the first time that uncontrolled influenza A virus replication actively suppresses MxA gene expression and emphasize the critical role of innate immunity in controlling influenza virus replication in vivo.
Immunoproteomic Discovery of Novel T Cell Antigens from the Obligate Intracellular PathogenChlamydia
Authors: Karunakaran Karuna P. , Rey-Ladino Jose , Stoynov Nikolay , Berg Kyra , Shen Caixia , Jiang Xiaozhou , Gabel Brent R. , Yu Hong , Foster Leonard J. , Brunham Robert C.
Published: April 2014

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-Ab) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.
Role of Galectin-3 in Leukocyte Recruitment in a Murine Model of Lung Infection by Streptococcus pneumoniae
Authors: Nieminen Julie , St-Pierre Christian , Bhaumik Pampa , Poirier Françoise , Sato Sachiko
Published: April 2014

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, β2-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a β2-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of β2-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces β2-integrin-independent migration.
An Important Role for Type III Interferon (IFN-λ/IL-28) in TLR-Induced Antiviral Activity
Authors: Ank Nina , Iversen Marie B. , Bartholdy Christina , Staeheli Peter , Hartmann Rune , Jensen Uffe B. , Dagnaes-Hansen Frederik , Thomsen Allan R. , Chen Zhi , Haugen Harald , Klucher Kevin , Paludan Søren R.
Published: April 2014

Type III IFNs (IFN-λ/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-λ was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Rα-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR−/−) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA−/− and IFNAR−/− mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-αβ expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-λ expression, whereas IL-28Rα signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Rα only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-λ and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.
Loss of Epithelial RelA Results in Deregulated Intestinal Proliferative/Apoptotic Homeostasis and Susceptibility to Inflammation
Authors: Steinbrecher Kris A. , Harmel-Laws Eleana , Sitcheran Raquel , Baldwin Albert S.
Published: April 2014

NF-κB plays a central, proinflammatory role in chronic intestinal inflammation, yet recent work suggests a predominantly protective function for this transcription factor group in some cell types of the intestine. We herein describe the conditional deletion of the NF-κB RelA gene in murine intestinal epithelia and determine its function in homeostatic control of enterocyte proliferation/apoptosis and susceptibility to colonic inflammation. Mice lacking RelA in ileal and colonic enterocytes were born in expected Mendelian ratios, and RelA-null epithelia differentiated normally. Spontaneous intestinal disease and death occurred with low penetrance in neonates lacking epithelial RelA. IκBα and IκBβ were significantly diminished in RelA-null epithelia, and endotoxin challenge revealed elevated p50 and c-Rel DNA binding activity as compared with controls. Deletion of RelA resulted in diminished expression of antimicrobial (defensin-related cryptdin 4, defensin-related cryptdin 5, RegIIIγ) and antiapoptotic, prorestitution genes (Bcl-xL, RegIV, IL-11, IL-18), and basal rates of epithelial apoptosis and proliferation were elevated. Mice lacking colonic RelA were sensitive to dextran sodium sulfate-induced colitis. Although experimental colitis enhanced proliferation in cells lacking RelA, sustained epithelial cell apoptosis precluded mucosal healing and decreased animal survival. We conclude that activation of RelA is required for homeostatic regulation of cell death and division in intestinal epithelia, as well as for protection from development of severe, acute inflammation of the intestine.
CD44 Is Critically Involved in Infarct Healing by Regulating the Inflammatory and Fibrotic Response
Authors: Huebener Peter , Abou-Khamis Tareq , Zymek Pawel , Bujak Marcin , Ying Xia , Chatila Khaled , Haudek Sandra , Thakker Geeta , Frangogiannis Nikolaos G.
Published: April 2014

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of “stop signals” that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44−/− animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44null infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44null cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-β in comparison to wild-type fibroblasts. The healing defects in CD44−/− mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.
Inhibitory ITAM Signaling by FcαRI-FcRγ Chain Controls Multiple Activating Responses and Prevents Renal Inflammation
Authors: Kanamaru Yutaka , Pfirsch Séverine , Aloulou Meryem , Vrtovsnik François , Essig Marie , Loirat Chantal , Deschênes Georges , Guérin-Marchand Claudine , Blank Ulrich , Monteiro Renato C.
Published: April 2014

Inhibitory signaling is an emerging function of ITAM-bearing immunoreceptors in the maintenance of homeostasis. Monovalent targeting of the IgA Fc receptor (FcαRI or CD89) by anti-FcαRI Fab triggers potent inhibitory ITAM (ITAMi) signaling through the associated FcRγ chain (FcαRI-FcRγ ITAMi) that prevents IgG phagocytosis and IgE-mediated asthma. It is not known whether FcαRI-FcRγ ITAMi signaling controls receptors that do not function through an ITAM and whether this inhibition requires Src homology protein 1 phosphatase. We show in this study that FcαRI-Fcγ ITAMi signals depend on Src homology protein 1 phosphatase to target multiple non-ITAM-bearing receptors such as chemotactic receptors, cytokine receptors, and TLRs. We found that anti-FcαRI Fab treatment in vivo reduced kidney inflammation in models of immune-mediated glomerulonephritis and nonimmune obstructive nephropathy by a mechanism that involved decreased inflammatory cell infiltration and fibrosis development. This treatment also prevented ex vivo LPS activation of monocytes from patients with lupus nephritis or vasculitis, as well as receptor activation through serum IgA complexes from IgA nephropathy patients. These findings point to a crucial role of FcαRI-FcRγ ITAMi signaling in the control of multiple heterologous or autologous inflammatory responses. They also identify anti-FcαRI Fab as a new potential therapeutic tool for preventing progression of renal inflammatory diseases.
Mechanisms for Decreased Function of B Cells in Aged Mice and Humans
Authors: Frasca Daniela , Landin Ana Marie , Riley Richard L. , Blomberg Bonnie B.
Published: April 2014

The immune system has been known for some time to be compromised in aged individuals, e.g., both mice and humans, and in both humoral and cellular responses. Our studies have begun to elucidate intrinsic B lymphocyte defects in Ig class switch recombination, activation-induced cytidine deaminase, and E47 transcription factor expression. These defects occur in both mice and humans. Our studies have also shown that tristetraprolin is one of the key players in regulating the decreased E47 mRNA stability in aged B lymphocytes. These and current studies should lead to improvements in B lymphocyte function in aged populations.
Mitigation of Experimental Allergic Encephalomyelitis by TGF-β Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance
Authors: Selvaraj Ramesh K. , Geiger Terrence L.
Published: April 2014

Stimulation of naive T lymphocytes in the presence of IL-2 and TGF-β induces the regulatory transcription factor Foxp3, which endows the cells with regulatory functions. To better understand the properties and therapeutic potential of these induced regulatory T cells (iTreg), we examined their immunomodulatory properties in myelin oligodendroglial glycoprotein-induced experimental allergic encephalomyelitis (MOG-EAE). Adoptively transferred iTreg were as potent as natural Foxp3+ Treg in preventing EAE development, and were active both prophylactically and after priming. The iTreg migrated into the CNS in quantity, skewing the ratio of regulatory to effector T lymphocytes. IL-10−/− iTreg failed to suppress disease, demonstrating a critical role for iTreg IL-10 production in their therapeutic activity. MOG-specific T cells from iTreg treated animals were anergic. The cells failed to proliferate in response to Ag except in the presence of exogenous IL-2, and did not secrete or secreted reduced amounts of IL-2, IFN-γ, and IL-17. MOG-specific T cells were not wholly unresponsive though, as they did secrete IL-10 after stimulation. To determine whether iTreg-mediated tolerance was infectious, fostering the development of T lymphocytes that could independently suppress EAE, we purged draining lymph node cells from MOG-immunized, iTreg treated mice of the administered iTreg, and transferred the remaining cells to Ag-inexperienced mice. The transferred cells were able to block EAE development. Thus iTreg are highly potent suppressors of autoimmune encephalomyelitis, and act in an IL-10 dependent manner both through the induction of anergy in effector T cells and through the infectious induction of protective T lymphocytes able to independently suppress disease development.
IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo
Authors: Kennedy Norton Sarah , Barnstein Brian , Brenzovich Jennifer , Bailey Daniel P. , Kashyap Mohit , Speiran Kelly , Ford Jill , Conrad Daniel , Watowich Stephanie , Moralle Matthew R. , Kepley Christopher L. , Murray Peter J. , Ryan John J.
Published: April 2014

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcεRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcεRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcεRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.
Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation
Authors: Costantino Cristina M. , Hang Howard C. , Kent Sally C. , Hafler David A. , Ploegh Hidde L.
Published: April 2014

Presentation of Ag by class II MHC is regulated by lysosomal proteases that not only destroy the class II invariant chain (Ii) chaperone but also generate the peptide Ag that is loaded onto the class II MHC dimer. We sought to determine the extent to which asparagine endopeptidase (AEP) influences human Ag and Ii processing. Our data confirm the constructive function of AEP in tetanus toxoid processing, but they are discordant with findings that suggest a destructive role for AEP in processing of the immunodominant myelin basic protein epitope. Furthermore, we observed no effect on invariant chain processing following AEP inhibition for several distinct allelic variants of human class II MHC products. We find that cysteine and aspartic proteases, as well as AEP, can act redundantly to initiate Ii processing. We detected considerable variation in lysosomal activity between different EBV-transformed B cell lines, but these differences do not result in altered regulation of invariant chain catabolism. We propose that, as for bound peptide Ag, the identity of the lysosomal enzyme that initiates invariant chain cleavage is dependent on the class II MHC allelic variants expressed.
The Role for Monocyte Chemoattractant Protein-1 in the Generation and Function of Memory CD8+ T Cells
Authors: Wang Tao , Dai Hehua , Wan Ni , Moore Yolonda , Dai Zhenhua
Published: April 2014

Memory T cells are resistant to the conventional costimulatory blockade and therefore impede tolerance induction. However, their migratory, survival, and functional requirements for chemokines are not well understood. We herein examine the role for MCP-1 or CCL2 in the generation, migration, and function of memory CD8+ T cells. We found that overall generation of both central memory (TCM) and effector memory (TEM) CD8+ T cells was severely impaired in the absence of MCP-1. Importantly, the survival of TEM, but not TCM, CD8+ cells was reduced without MCP-1, whereas the homeostatic proliferation of TCM, but not TEM, CD8+ cells was weakened in MCP-1−/− mice. However, once they were generated in the absence of MCP-1, in vitro function of both subsets of memory cells remained intact as determined by their proliferation and IFN-γ production. Interestingly, the migration of TCM, but not TEM, CD8+ cells to inflammatory sites was significantly delayed without MCP-1, whereas both subsets of memory cells underwent comparable expansion and apoptosis with or without MCP-1 during the effector phase. Moreover, the function to eliminate a graft of TCM, but not TEM, CD8+ cells was impaired without MCP-1. Thus, this study demonstrates that MCP-1 plays an important role in not only migration but also generation and survival of memory T cells. This finding provides new insight into the requirement of chemokines for the generation, survival, and function of differential subsets of memory T cells and may have clinic implications for tolerance induction.