bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival
Authors: Chen Hui-Chen , Byrd John C. , Muthusamy Natarajan
Published: April 2014

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser119-ala phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed ∼70% increase in pre-B1 (CD43+B220+CD24+(int)) and ∼60% decreased pre-BII (CD43+B220+CD24++(high)) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed ∼4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2−/− mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43+B220+CD24high population that is normally absent in RAG-2−/− mice. In the periphery, the Tg mice exhibited decreased CD21dimCD23highIgM+ follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.
The JNK Are Important for Development and Survival of Macrophages
Authors: Himes S. Roy , Sester David P. , Ravasi Timothy , Cronau Stephen L. , Sasmono Tedjo , Hume David A.
Published: April 2014

We report in this study that activation of the JNK by the growth factor, CSF-1 is critical for macrophage development, proliferation, and survival. Inhibition of JNK with two distinct classes of inhibitors, the pharmacological agent SP600125, or the peptide D-JNKI1 resulted in cell cycle inhibition with an arrest at the G2/M transition and subsequent apoptosis. JNK inhibition resulted in decreased expression of CSF-1R (c-fms) and Bcl-xL mRNA in mature macrophages and repressed CSF-1-dependent differentiation of bone marrow cells to macrophages. Macrophage sensitivity to JNK inhibitors may be linked to phosphorylation of the PU.1 transcription factor. Inhibition of JNK disrupted PU.1 binding to an element in the c-fms gene promoter and decreased promoter activity. Promoter activity could be restored by overexpression of PU.1. A comparison of expression profiles of macrophages with 22 other tissue types showed that genes that signal JNK activation downstream of tyrosine kinase receptors, such as focal adhesion kinase, Nck-interacting kinase, and Rac1 and scaffold proteins are highly expressed in macrophages relative to other tissues. This pattern of expression may underlie the novel role of JNK in macrophages.
Mast Cells Enhance T Cell Activation: Importance of Mast Cell Costimulatory Molecules and Secreted TNF
Authors: Nakae Susumu , Suto Hajime , Iikura Motoyasu , Kakurai Maki , Sedgwick Jonathon D. , Tsai Mindy , Galli Stephen J.
Published: April 2014

We recently reported that mast cells stimulated via FcεRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3+ T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.
A Polymeric Bacterial Protein Activates Dendritic Cells via TLR4
Authors: Berguer Paula M. , Mundiñano Juliana , Piazzon Isabel , Goldbaum Fernando A.
Published: April 2014

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.
Meningococcal Porin PorB Binds to TLR2 and Requires TLR1 for Signaling
Authors: Massari Paola , Visintin Alberto , Gunawardana Jay , Halmen Kristen A. , King Carol A. , Golenbock Douglas T. , Wetzler Lee M.
Published: April 2014

TLR2 plays a key role in the initiation of the cellular innate immune responses by a wide range of bacterial products. TLRs signaling, including TLR2 and its coreceptors TLR1 and TLR6, is mediated by a number of specific ligands. Although many of the TLR-mediated cell signaling pathways have been elucidated in the past few years, the molecular mechanisms that lead to cell activation are still poorly understood. In this study, we investigate the interaction of PorB from Neisseria meningitidis with TLR2 and describe the direct binding of a bacterial protein to TLR2 for the first time. Using labeled PorB, we demonstrate its binding to TLR2 both in its soluble form in vitro, and when it is over-expressed on the surface of human embryonic kidney 293 cells. We also show that TLR2-mediated binding of PorB is directly related to cellular activation. In addition, using 293 cells expressing the chimeric TLR2/TLR1 and TLR2/TLR6 complexes, we report the selectivity of PorB binding to the TLR2/TLR1 heterodimer, which is required for initiating signaling in transfected 293 cells and in murine B cells. Together, these data provide new evidence that TLR2 recognizes PorB through direct binding, and that PorB-induced cell activation is mediated by a TLR2/TLR1 complex.
Endotoxin-Induced Down-Regulation of Elk-3 Facilitates Heme Oxygenase-1 Induction in Macrophages
Authors: Chung Su Wol , Chen Yen-Hsu , Yet Shaw-Fang , Layne Matthew D. , Perrella Mark A.
Published: April 2014

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that is acutely induced by inflammatory stimuli, and the products of HO-1-mediated heme degradation have anti-inflammatory properties. In many different pathophysiologic states, the up-regulation of HO-1 has been shown to be beneficial in combating the detrimental consequences of increased inflammation. Ets transcription factors are known to be important mediators of inflammatory responses, and the ternary complex factor subfamily of Ets proteins has both transcriptional activation and repression activity. The present study demonstrates that of several ternary complex factor subfamily members, only Elk-3 represses HO-1 promoter activity in macrophages. Endotoxin administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this reduction in Elk-3 preceded the LPS-mediated up-regulation of HO-1 message. Analogous results also occurred in lung tissue of mice exposed to endotoxin. Two putative Ets binding sites (EBS1 and EBS2) are present in the downstream region of the murine HO-1 promoter (bp −125 and −93, respectively), and we recently showed that the EBS2 site is essential for HO-1 induction by endotoxin. In contrast, the present study demonstrates that the repressive effect of Elk-3 on HO-1 promoter activity is dependent on the EBS1 site. Taken together, our data reveal that Elk-3 serves as an important repressor of HO-1 gene transcription and contributes to the tight control of HO-1 gene regulation in the setting of inflammatory stimuli.
OX40 Ligation of CD4+ T Cells Enhances Virus-Specific CD8+ T Cell Memory Responses Independently of IL-2 and CD4+ T Regulatory Cell Inhibition
Authors: Yu Qigui , Yue Feng Yun , Gu Xiao X. , Schwartz Herbert , Kovacs Colin M. , Ostrowski Mario A.
Published: April 2014

We have previously shown that CD4+ T cells are required to optimally expand viral-specific memory CD8+ CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4+ T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4+ T cells, which subsequently can help CD8+ T cell responses. The current study examined whether OX40 ligation on ex vivo CD4+ T cells can enhance their ability to “help” virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4+ T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4+ T regulatory cells, but was associated with a direct effect on proliferation of CD4+ T cells. Thus, OX40 ligation on CD4+ T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.
Impaired Protection against Mycobacterium bovis Bacillus Calmette-Guérin Infection in IL-15-Deficient Mice
Authors: Saito Kimika , Yajima Toshiki , Kumabe Shino , Doi Takehiko , Yamada Hisakata , Sad Subash , Shen Hao , Yoshikai Yasunobu
Published: April 2014

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15−/−) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15−/− mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4+ Th1 response capable of producing IFN-γ was normally detected in spleen and lung of IL-15−/− mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-γ and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15−/− mice. The degree of proliferation of Ag-specific CD8+ T cells in IL-15−/− mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8+ T cells significantly increased in IL-15−/− mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.
Gliadin Stimulation of Murine Macrophage Inflammatory Gene Expression and Intestinal Permeability Are MyD88-Dependent: Role of the Innate Immune Response in Celiac Disease
Authors: Thomas Karen E. , Sapone Anna , Fasano Alessio , Vogel Stefanie N.
Published: April 2014

Recent studies have demonstrated the importance of TLR signaling in intestinal homeostasis. Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. In this study, we sought to test the hypothesis that gliadin initiates this response by stimulating the innate immune response to increase intestinal permeability and by up-regulating macrophage proinflammatory gene expression and cytokine production. To this end, intestinal permeability and the release of zonulin (an endogenous mediator of gut permeability) in vitro, as well as proinflammatory gene expression and cytokine release by primary murine macrophage cultures, were measured. Gliadin and its peptide derivatives, 33-mer and p31-43, were found to be potent inducers of both a zonulin-dependent increase in intestinal permeability and macrophage proinflammatory gene expression and cytokine secretion. Gliadin-induced zonulin release, increased intestinal permeability, and cytokine production were dependent on myeloid differentiation factor 88 (MyD88), a key adapter molecule in the TLR/IL-1R signaling pathways, but were neither TLR2- nor TLR4-dependent. Our data support the following model for the innate immune response to gliadin in the initiation of CD. Gliadin interaction with the intestinal epithelium increases intestinal permeability through the MyD88-dependent release of zonulin that, in turn, enables paracellular translocation of gliadin and its subsequent interaction with macrophages within the intestinal submucosa. There, the interaction of gliadin with macrophages elicits a MyD88-dependent proinflammatory cytokine milieu that facilitates the interaction of T cells with APCs, leading ultimately to the Ag-specific adaptive immune response seen in patients with CD.
Proinflammatory Cytokine Production in Liver Regeneration Is Myd88-Dependent, but Independent of Cd14, Tlr2, and Tlr4
Authors: Campbell Jean S. , Riehle Kimberly J. , Brooling John T. , Bauer Renay L. , Mitchell Claudia , Fausto Nelson
Published: April 2014

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.
Priming of Glomerular Mesangial Cells by Activated Macrophages Causes Blunted Responses to Proinflammatory Stimuli
Authors: Hayakawa Kunihiro , Meng Yiman , Hiramatsu Nobuhiko , Kasai Ayumi , Yamauchi Kozue , Yao Jian , Kitamura Masanori
Published: April 2014

Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. Activated macrophages trigger mesangial cells to express an array of inflammation-associated genes via activation of NF-κB and AP-1. However, this inflammatory response is often transient and subsides spontaneously. We found that mesangial cells activated by bystander macrophages showed blunted responses of NF-κB to subsequent macrophage exposure. It was associated with sustained levels of IκBβ, but not IκBα. The tolerance observed was reversible and reproduced by conditioned media from activated macrophages (macrophage-conditioned medium (MφCM)). In vivo priming of mesangial cells by activated glomerular macrophages also caused the tolerance of mesangial cells. The macrophage-derived tolerance inducers were heat-labile, and multiple molecules were involved. Among inflammatory cytokines produced by macrophages, TNF-α and IL-1β were able to induce mesangial cell tolerance dose-dependently. The mesangial cell tolerance was also observed in activation of the MAPK-AP-1 pathway; i.e., phosphorylation of ERK, JNK, and p38 MAPK by macrophages was blunted when the cells were pre-exposed to MφCM. Induction of c-fos and c-jun was also abrogated in mesangial cells pre-exposed to MφCM, and the suppression was attenuated by blockade of MAPK activation during the first exposure to MφCM. These data elucidated that mesangial cells, once exposed to macrophages, become insensitive to subsequent activation by macrophages and proinflammatory stimuli. This self defense of glomerular cells may play a role in the resolution of macrophage-mediated, acute glomerulonephritis.
Impaired GATA3-Dependent Chromatin Remodeling and Th2 Cell Differentiation Leading to Attenuated Allergic Airway Inflammation in Aging Mice
Authors: Hasegawa Akihiro , Miki Takako , Hosokawa Hiroyuki , Hossain Mohammad B. , Shimizu Chiori , Hashimoto Kahoko , Kimura Motoko Y. , Yamashita Masakatsu , Nakayama Toshinori
Published: April 2014

Age-related changes in lymphocytes are most prominent in the T cell compartment. There have been substantial numbers of reports on T cell function in aged mice and humans, such as on the production of Th1 and Th2 cytokines, but the results show considerable variation and contradictions. In the present study, we used 8- to 12-mo-old aging mice and a well-established in vitro Th1/Th2 cell differentiation culture system to identify molecular defects in Th1/Th2 cell differentiation that can be detected in the relatively early stages of aging. The capability to differentiate into Th2 cells is reduced in aging mouse CD4+ T cells. Decreased activation of the ERK MAPK cascade upon TCR stimulation, but normal intracellular-free calcium ion concentration mobilization and normal IL-4-induced STAT6 activation were observed in aging mouse CD4+ T cells. In addition, reduced expression of GATA3 was detected in developing Th2 cells. Chromatin remodeling of the Th2 cytokine gene locus was found to be impaired. Th2-dependent allergic airway inflammation was milder in aging mice compared with in young adult mice. These results suggest that the levels of Th2 cell differentiation and resulting Th2-dependent immune responses, including allergic airway inflammation, decline during aging through defects in the activation of the ERK MAPK cascade, expression of GATA3 protein and GATA3-dependent chromatin remodeling of the Th2 cytokine gene locus. In the present study, we provide the first evidence indicating that a chromatin-remodeling event in T cells is impaired by aging.
HIV-1 Envelope Triggers Polyclonal Ig Class Switch Recombination through a CD40-Independent Mechanism Involving BAFF and C-Type Lectin Receptors
Authors: He Bing , Qiao Xugang , Klasse Per J. , Chiu April , Chadburn Amy , Knowles Daniel M. , Moore John P. , Cerutti Andrea
Published: April 2014

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4+ T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4+ T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4+ T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.
Distinct, Cross-Reactive Epitope Specificities of CD8 T Cell Responses Are Induced by Natural Hepatitis B Surface Antigen Variants of Different Hepatitis B Virus Genotypes
Authors: Riedl Petra , Bertoletti Antonio , Lopes Ross , Lemonnier Francois , Reimann Jörg , Schirmbeck Reinhold
Published: April 2014

We investigated the specific and cross-reactive CD8 T cell immunity to three natural variants (of different geno/serotype) of the small hepatitis B surface Ag (or S protein). The Dd-binding variants of the S201–209 epitope showed different immunogenicity. The loss of the consensus C-terminal (P9) anchor abrogated its immunogenicity. In contrast, a conservative (serine vs asparagine) exchange at P7 primed cross-reactive CD8 T cells that preferentially recognized the priming variant. Cross-reactive CD8 T cell responses to a variant could be primed in mice tolerant to an alternative variant of the Dd-binding S201–209 peptide. Loss of the C-terminal (P10) anchor in S185–194 eliminated its immunogenicity in HLA-A*0201(A2)-transgenic mice but two conservative exchanges (leucine vs valine in P2, and leucine vs isoleucine in P6) in S208–216 generated cross-reactive CD8 T cell responses with strong preference for the priming variant. Similar cross-reactive recognition of variant envelope epitopes were also found in S208–216-specific CD8 T cells from hepatitis B virus (HBV)-infected patients. Distinct CD8 T cell populations cross-reactive to natural variants of class I-restricted HBV epitopes can be primed by vaccination (of mice) or natural infection (of humans), and they may play a role in the “spontaneous remission” or the specific immunotherapy of chronic HBV infection.
Long-Term Retention of Mature NK1.1+ NKT Cells in the Thymus
Authors: Berzins Stuart P. , McNab Finlay W. , Jones Claerwen M. , Smyth Mark J. , Godfrey Dale I.
Published: April 2014

The NKT cell pool in the thymus contains immature (NK1.1−) and mature (NK1.1+) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1− NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1+ NKT cells. In this study we have determined that mature NK1.1+ NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-γ and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1− cells despite mature NK1.1+ NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.
Immature NK Cells Suppress Dendritic Cell Functions during the Development of Leukemia in a Mouse Model
Authors: Ebata Kazumi , Shimizu Yukihiro , Nakayama Yasuhiro , Minemura Masami , Murakami Jun , Kato Tsutomu , Yasumura Satoshi , Takahara Terumi , Sugiyama Toshiro , Saito Shigeru
Published: April 2014

To analyze the mechanisms by which cancer cells escape from hosts’ immune surveillance, we investigated the changes in immune status during the progression of leukemia induced by injecting mice with WEHI-3B cells. In the bone marrow (BM) of leukemic mice, only DX5+CD3− cells were continuously increased, despite the progression of leukemia. In addition, DX5+CD3− cells were rapidly increased in peripheral blood (PB) 20 days after inoculation. We also found that myeloid dendritic cells (DCs) expressing low levels of I-Ad and having low allo-T cell stimulatory activity were markedly increased in PB and spleen. The increase in DX5+ cells in BM was thought to be induced by soluble factors from leukemic cells. DX5+ cells from leukemic mice were CD3−, B220−, Gr-1−, CD14−, CD94−, Ly-49C/F−, asialo GM1+, CD25+, CD122+, Thy-1bright, and c-kitdim and showed low killing activity against YAC-1 cells, suggesting that those DX5+ cells were immature NK cells. NK cells from leukemic PB down-regulated the expression of I-Ad on DCs, an effect mediated by TGF-β. Moreover, these NK cells significantly suppressed the allo-T cell stimulatory activity of DCs, an effect requiring cell-to-cell contact between NK cells and DCs and thought to involve CD25. Importantly, NK cells from leukemic PB inhibited generation of autotumor-specific CTL induced by DCs in primary MLR or by DC immunization. In conclusion, we identified circulating immature NK cells with immunosuppressive activities. These cells may be important for understanding the involvement of the host immune system during the development of leukemia.
TLR3-Involved Modulation of Pregnancy Tolerance in Double-Stranded RNA-Stimulated NOD/SCID Mice
Authors: Lin Yi , Liang Zhenhong , Chen Yijing , Zeng Yaoying
Published: April 2014

This study aims to extend understanding of the relationship between TLR3-involved cell signaling and dsRNA-induced embryo resorption. Upon stimulation of dsRNA, the resorption rate of embryos was boosted dramatically in syngeneic mating BALB/c mice, but not significantly influenced in syngeneic mating NOD/SCID mice. Accordingly, there was an enhanced cell surface expression of TLR3 on placental CD45+ cells derived from BALB/c mice, concomitant with both increased percentages of CD45+CD80+ cells and CD8α+CD80+ cells in flow cytometric analysis. In addition, both increased IL-2 and decreased IL-10 expression could be observed in CD45+ cell group in the intracellular detection by flow cytometry. In contrast, no such trends were observed in NOD/SCID model, and its resorption rate of embryos was kept at a low level throughout pregnancy. Neutralizing Abs against TLR3 could abrogate the embryo rejection induced by dsRNA in BALB/c mice, and simultaneously could reduce the CD80+ percentage in the CD45+ cell group. These results indicate that the interaction between dsRNA and TLR3 may be involved in the mobilization of CD45+CD80+ and CD8α+CD80+ cells, followed by the up-regulation of IL-2 and down-regulation of IL-10 expression at the feto-maternal interface, and finally resulting in embryo rejection. The relatively low responsiveness of NOD/SCID mice may be one of the reasons why these mice appeared to be resistant to dsRNA-induced embryo resorption.
Relationships between Distinct Blood Monocyte Subsets and Migrating Intestinal Lymph Dendritic Cells In Vivo under Steady-State Conditions
Authors: Yrlid Ulf , Jenkins Christopher D. , MacPherson G. Gordon
Published: April 2014

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (MOs), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of MOs, defined by expression of different chemokine receptors, CCR2 and CX3CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2highCX3CR1low MOs but have not defined unambiguously the origin and fate of CCR2lowCX3CR1high cells. In this study, we show that rat MOs can also be divided into CCR2highCX3CR1low(CD43low) and CCR2lowCX3CR1high(CD43high) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain MOs, including the marginating pool, we show by adoptive transfer that CD43low MOs can differentiate into CD43high MOs in blood without cell division. By adoptive transfer of blood MOs followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2lowCX3CR1high(CD43high) blood MOs in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2lowCX3CR1high blood MOs and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli.
Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region
Authors: Johnston Colette M. , Wood Andrew L. , Bolland Daniel J. , Corcoran Anne E.
Published: April 2014

The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5′ end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3′ end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.
Agonistic Antibody to TLR4/MD-2 Protects Mice from Acute Lethal Hepatitis Induced by TNF-α
Authors: Akashi-Takamura Sachiko , Furuta Takahisa , Takahashi Koichiro , Tanimura Natsuko , Kusumoto Yutaka , Kobayashi Toshihiko , Saitoh Shin-ichiroh , Adachi Yoshiyuki , Doi Takahiro , Miyake Kensuke
Published: April 2014

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-α, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-κB through TLR4/MD-2.
Hemin Activation Ameliorates HIV-1 Infection via Heme Oxygenase-1 Induction
Authors: Devadas Krishnakumar , Dhawan Subhash
Published: April 2014

Hemin, a critical component of hemoglobin, is an active ingredient of a biologic therapeutic approved by the Food and Drug Administration for the treatment of acute porphyries. This report describes a biological function of this molecule in inducing host defense against HIV-1 infection via heme oxygenase-1 (HO-1) induction. Treatment of monocytes with hemin substantially inhibited HIV replication, as evident by nearly undetectable viral RNA and cell-free HIV-1 p24 protein in a dose-dependent manner. Hemin exposure of these cells before infection, at the time of infection, or after infection caused >90% reduction of HIV DNA with substantially low levels of HIV-1 p24 and HIV-associated cytopathic effects. In addition, hemin treatment significantly suppressed infection of both monocytes and T cells inoculated with R5, X4, R5X4 tropic strains, and reverse transcriptase-resistant, azidothymidine-resistant, ddC/ddI-resistant, nivirapine-resistant, and other clinical HIV isolates. Intraperitoneal administration of hemin 4 days after HIV infection reduced viral load in the serum of human PBMC-reconstituted nonobese diabetic SCID mice by >6-fold. Suppression of HIV replication in hemin-activated cells correlated with the induction of HO-1 and was attenuated by tin protoporphyrin (SnPP) IX, an inhibitor of HO-1 activity, suggesting a pivotal role of this endogenous enzyme in the regulation of HIV infection. Hemin-induced HO-1 induction in the CCR-5, CXCR-4, and CD4 coexpressing GHOST(3) cells was consistent with the inhibition of Tat-dependent activation of long terminal repeat promoter leading to reduced GFP expression. These findings suggest an important role of hemin-induced HO-1 activity as a host defense mechanism against HIV-1 infection.
Homing and Function of Human Skin γδ T Cells and NK Cells: Relevance for Tumor Surveillance
Authors: Ebert Lisa M. , Meuter Simone , Moser Bernhard
Published: April 2014

Normal (noninflamed) human skin contains a network of lymphocytes, but little is known about the homing and function of these cells. The majority of αβ T cells in normal skin express CCR8 and produce proinflammatory cytokines. In this study we examined other subsets of cutaneous lymphocytes, focusing on those with potential function in purging healthy tissue of transformed and stressed cells. Human dermal cell suspensions contained significant populations of Vδ1+ γδ T cells and CD56+CD16− NK cells, but lacked the subsets of Vδ2+ γδ T cells and CD56+CD16+ NK cells, which predominate in peripheral blood. The skin-homing receptors CCR8 and CLA were expressed by a large fraction of both cell types, whereas chemokine receptors associated with lymphocyte migration to inflamed skin were absent. Neither cell type expressed CCR7, although γδ T cells up-regulated this lymph node-homing receptor upon TCR triggering. Stimulation of cutaneous Vδ1+ γδ T cell lines induced secretion of large amounts of TNF-α, IFN-γ, and the CCR8 ligand CCL1. In contrast to cutaneous αβ T cells, both cell types had the capacity to produce intracellular perforin and displayed strong cytotoxic activity against melanoma cells. We therefore propose that γδ T cells and NK cells are regular constituents of normal human skin with potential function in the clearance of tumor and otherwise stressed tissue cells.
Lung-Restricted Macrophage Activation in the Pearl Mouse Model of Hermansky-Pudlak Syndrome
Authors: Young Lisa R. , Borchers Michael T. , Allen Holly L. , Gibbons Reta S. , McCormack Francis X.
Published: April 2014

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring “pearl” HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1γ) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-α, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-α, MIP1α, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-α responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-γ/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-α at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-α secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.
Critical Role for Mast Cells in the Pathogenesis of 2,4-Dinitrobenzene-Induced Murine Colonic Hypersensitivity Reaction
Authors: Rijnierse Anneke , Koster Andries S. , Nijkamp Frans P. , Kraneveld Aletta D.
Published: April 2014

The immunological mechanisms underlying the role of mast cells in the pathogenesis of inflammatory bowel disease (IBD) are poorly defined. In this study, non-IgE mediated colonic hypersensitivity responses in BALB/c mice induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dinitrobenzene sulfonic acid featured as a model to study the role of mast cells in the development of IBD. Vehicle- or DNFB-sensitized mice were monitored for clinical symptoms and inflammation 72 h after dinitrobenzene sulfonic acid challenge. DNFB-sensitized mice developed diarrheic stool, increased colonic vascular permeability, hypertrophy of colonic lymphoid follicles (colonic patches), and showed cellular infiltration at the microscopic level. Increased numbers of mast cells were found in the colon of DNFB-sensitized mice located in and around colonic patches associated with elevated levels of mouse mast cell protease-1 in plasma indicating mast cell activation. Colonic patches of DNFB mice, stimulated in vitro with stem cell factor indicated that an increase in TNF-α levels in the colon is mainly mast cell originated. Finally, neutrophil infiltration was observed in the colon of DNFB-sensitized mice. Induction of this model in mast cell-deficient WBB6F1 W/Wv mice shows a profound reduction of characteristics of the colonic hypersensitivity reaction. Reconstitution with bone marrow-derived mast cells in WBB6F1 W/Wv mice fully restored the inflammatory response. This study demonstrates the importance of mast cells in the development of clinical symptoms and inflammation in the presented murine model for IBD.
Severe Disease, Unaltered Leukocyte Migration, and Reduced IFN-γ Production in CXCR3−/− Mice with Experimental Autoimmune Encephalomyelitis
Authors: Liu LiPing , Huang DeRen , Matsui Masaru , He Toby T. , Hu Taofang , DeMartino Julie , Lu Bao , Gerard Craig , Ransohoff Richard M.
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3−/− mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3+/+) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3+/+ and CXCR3−/− mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3−/− mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3+/+ mice. Spinal cords of CXCR3−/− mice with EAE demonstrated decreased levels of IFN-γ, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3−/− mice primed with MOG35–55 secreted less IFN-γ in Ag-driven recall responses than cells from CXCR3+/+ animals. CXCR3−/− lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-γ. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-γ production and downstream events that affect disease severity.
The Vast Majority of CLA+ T Cells Are Resident in Normal Skin
Authors: Clark Rachael A. , Chong Benjamin , Mirchandani Nina , Brinster Nooshin K. , Yamanaka Kei-ichi , Dowgiert Rebecca K. , Kupper Thomas S.
Published: April 2014

There are T cells within normal, noninflamed skin that most likely conduct immunosurveillance and are implicated in the development of psoriasis. We isolated T cells from normal human skin using both established and novel methods. Skin resident T cells expressed high levels of CLA, CCR4, and CCR6, and a subset expressed CCR8 and CXCR6. Skin T cells had a remarkably diverse TCR repertoire and were mostly Th1 memory effector cells with smaller subsets of central memory, Th2, and functional T regulatory cells. We isolated a surprising number of nonexpanded T cells from normal skin. To validate this finding, we counted T cells in sections of normal skin and determined that there are ∼1 × 106 T cells/cm2 normal skin and an estimated 2 × 1010 T cells in the entire skin surface, nearly twice the number of T cells in the circulation. Moreover, we estimate that 98% of CLA+ effector memory T cells are resident in normal skin under resting conditions. These findings demonstrate that there is a large pool of memory T cells in normal skin that can initiate and perpetuate immune reactions in the absence of T cell recruitment from the blood.
Iodination of Tyrosyls in Thyroglobulin Generates Neoantigenic Determinants That Cause Thyroiditis
Authors: Li Haiyan S. , Carayanniotis George
Published: April 2014

Thyroglobulin (Tg) is unique in its ability to incorporate and store available iodine in the form of iodotyrosyl residues. Iodination of Tg has been known to increase its immunopathogenicity in experimental animals, presumably through the formation of iodine-containing neoantigenic determinants that can elicit an autoimmune response, but defined pathogenic Tg peptides carrying iodotyrosyls have not yet been identified. We report in this study that a systematic, algorithm-based search of mouse Tg has delineated three iodotyrosyl-containing peptides that activate autoreactive T cells and cause experimental autoimmune thyroiditis in normal CBA/J mice. These peptides (aa 117–132, 304–318, and 1931–1945) were not immunogenic in their native form, and iodination of tyrosyls facilitated either peptide binding to MHC or T cell recognition of the peptide. These results demonstrate that iodotyrosyl formation in normal Tg confers pathogenic potential to certain peptides that may otherwise remain innocuous and undetectable by conventional mapping methods.
The Multiple Personalities of the Chemokine Receptor CCR7 in Dendritic Cells
Authors: Sánchez-Sánchez Noelia , Riol-Blanco Lorena , Rodríguez-Fernández José Luis
Published: April 2014

CCR7 was described initially as a potent leukocyte chemotactic receptor that was later shown to be responsible of directing the migration of dendritic cells (DCs) to the lymph nodes where these cells play an important role in the initiation of the immune response. Recently, a variety of reports have indicated that, apart from chemotaxis, CCR7 controls the cytoarchitecture, the rate of endocytosis, the survival, the migratory speed, and the maturation of the DCs. Some of these functions of CCR7 and additional ones also have been described in other cell types. Herein we discuss how this receptor may contribute to modulate the immune response by regulating different functions in DCs. Finally, we also suggest a possible mechanism whereby CCR7 may control its multiple tasks in these cells.
Selection of Anti-Double-Stranded DNA B Cells in Autoimmune MRL-lpr/lprMice
Authors: Chen Ching , Li Hui , Tian Qi , Beardall Michael , Xu Yang , Casanova Nina , Weigert Martin
Published: April 2014

Abs to DNA and nucleoproteins are expressed in systemic autoimmune diseases, whereas B cells producing such Abs are edited, deleted, or inactivated in healthy individuals. Why autoimmune individuals fail to regulate is not well understood. In this study, we investigate the sources of anti-dsDNA B cells in autoimmune transgenic MRL-lpr/lpr mice. These mice are particularly susceptible to lupus because they carry a site-directed transgene, H76R that codes for an anti-DNA H chain. Over 90% of the B cells are eliminated in the bone marrow of these mice, and the few surviving B cells are associated with one of two Vκ editors, Vκ38c and Vκ21D. Thus, it appears that negative selection by deletion and editing are intact in MRL-lpr/lpr mice. However, a population of splenic B cells in the H76R MRL-lpr/lpr mice produces IgG anti-nuclear Abs, and these mice have severe autoimmune organ damage. These IgG Abs are not associated with editors but instead use a unique Vκ gene, Vκ23. The H76R/Vκ23 combination has a relatively high affinity for dsDNA and an anti-nuclear Ab pattern characteristic of lupus. Therefore, this Vκ gene may confer a selective advantage to anti-DNA Abs in diseased mice.
Regulation of Intestinal Dendritic Cell Migration and Activation by Plasmacytoid Dendritic Cells, TNF-α and Type 1 IFNs after Feeding a TLR7/8 Ligand
Authors: Yrlid Ulf , Milling Simon W. F. , Miller Joanna L. , Cartland Sian , Jenkins Christopher D. , MacPherson G. Gordon
Published: April 2014

Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-α dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.