bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-Inflammatory Effects of FTY720 against Viral-Induced Immunopathology: Role of Drug-Induced Conversion of T Cells to Become Foxp3+ Regulators
Authors: Sehrawat Sharvan , Rouse Barry T.
Published: April 2014

FTY720 has been used to control inflammatory lesions, but the mechanisms by which the drug acts in vivo are poorly understood. Such mechanisms may result primarily from effects on lymphocyte and dendritic cell homing to lymphoid and inflammatory sites. We demonstrate that FTY720 may also act by causing the conversion of TCR-stimulated nonregulatory CD4+ T cells to Foxp3+CD4+ regulatory T cells and by enhancing their suppressive activity. In a model in which mice were ocularly infected with HSV, daily treatment with FTY720 resulted in significantly diminished ocular lesions. The treated animals showed increased frequencies of Foxp3+ T cells in lymphoid organs and at two inflammatory sites, namely cornea and trigeminal ganglia. In a second series of experiments, immunized DO11.10RAG2−/− animals, normally lacking endogenous Foxp3+ T cells, that were given FTY720 treatment developed high frequencies of Foxp3+ regulatory T cells in lymph nodes. Some converted cells persisted in treated animals for several weeks after drug administration was discontinued. Finally, FTY720 could effectively induce Foxp3-expressing cells from Foxp3− cells in vitro, an effect inhibited by anti-TGF-β or the proinflammatory cytokine IL-6. Accordingly, the anti-inflammatory effects of FTY720 could be mediated at least in part by its ability to cause the conversion of Ag-stimulated conventional T cells to become Foxp3+ regulators. The use of FTY720 along with Ag administration could represent a useful therapeutic means to selectively expand Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.
Nicotine Primarily Suppresses Lung Th2 but Not Goblet Cell and Muscle Cell Responses to Allergens
Authors: Mishra Neerad C. , Rir-sima-ah Jules , Langley Raymond J. , Singh Shashi P. , Peña-Philippides Juan C. , Koga Takeshi , Razani-Boroujerdi Seddigheh , Hutt Julie , Campen Matthew , Kim K. Chul , Tesfaigzi Yohannes , Sopori Mohan L.
Published: April 2014

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.
Preferential In Situ CD4+CD56+ T Cell Activation and Expansion within Human Glioblastoma
Authors: Waziri Allen , Killory Brendan , Ogden Alfred T. , Canoll Peter , Anderson Richard C. E. , Kent Sally C. , Anderson David E. , Bruce Jeffrey N.
Published: April 2014

Recent evidence suggests that suppression of the cellular immune response is often attributable to populations of functionally distinct T cells that act to down-regulate Ag-specific effector T cells. Using flow cytometry, we evaluated tumor-infiltrating lymphocytes (TIL) from patients undergoing neurosurgical resection of glioblastoma multiforme (GBM), metastatic lung carcinoma, and meningioma for markers known to be expressed on immunoregulatory T cells. Ex vivo phenotypic characteristics, cellular proliferation, and cytokine expression patterns were compared between T cell subsets found in the PBMC and within TIL from fresh tumor samples. Interestingly, nearly half of all T cells infiltrating GBM specimens were CD56+ T cells, while much smaller percentages of similar cells were identified within metastatic lung tumors and meningiomas. CD56+ T cells identified within GBM were not canonical, or “invariant,” NKT cells, as they demonstrated diverse TCR expression, a primarily CD4 single-positive phenotype, and lack of CD1d reactivity. The percentage of CD56+ T cells exhibiting evidence of proliferation within GBM was 3- to 4-fold higher than the proportion of proliferating CD56− T cells from these lesions. In addition, direct ex vivo analysis of cytokine expression by TIL from GBM demonstrated significant numbers of IL-4/IL-13 positive cells, cytokines that are integral in the cell-mediated repression of tumor immunity in experimental models. We propose that GBM has a unique capacity to recruit and activate CD4+CD56+ T cells, a population that has not been previously described within human tumors.
Local Accumulation of FOXP3+ Regulatory T Cells: Evidence for an Immune Evasion Mechanism in Patients with Large Condylomata Acuminata
Authors: Cao Yuchun , Zhao Jie , Lei Zhang , Shen Shiqian , Liu Cong , Li Dong , Liu Jihong , Shen Guan-Xin , Zhang Gui-Mei , Feng Zuo-Hua , Huang Bo
Published: April 2014

Condylomata acuminata derived from the infection of human papillomavirus is a common sexually transmitted disease. Although T cell-mediated cellular immunity is considered as the main arm against such infection, the regulation of T cell immune responses in genital condylomata is unclear to date. In this study, we analyzed FOXP3+ regulatory T cells in genital condylomata of patients. The results show that FOXP3+ regulatory T cells with suppressive function accumulated in large warts. Consistently, the immunosuppressive milieu in large warts was characterized by high expression of IL-10 and TGF-β1 and low expression of IL-2 and IFN-γ. The responsiveness of wart-infiltrating T cells both in vitro and in vivo can be increased by depleting FOXP3+ T cells. The accumulation of FOXP3+ regulatory T cells in large warts can be partly ascribed to the chemotaxis of CCL17 and CCL22, derived from Langerhans cells and macrophages in wart. Although such accumulation favors the local immunosuppression, it seems not to influence the systemic immunity. In conclusion, these findings demonstrate that FOXP3+ regulatory T cells play an important role in genital condylomata, which has multiple implications in the comprehensive treatment of condylomata acuminata.
No Advantage of Cell-Penetrating Peptides over Receptor-Specific Antibodies in Targeting Antigen to Human Dendritic Cells for Cross-Presentation
Authors: Tacken Paul J. , Joosten Ben , Reddy Anita , Wu Dayang , Eek Annemarie , Laverman Peter , Kretz-Rommel Anke , Adema Gosse J. , Torensma Ruurd , Figdor Carl G.
Published: April 2014

Induction of CTL responses by dendritic cell (DC)-based vaccines requires efficient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-specific Abs improves Ag loading of DCs. In contrast to CPPs, receptor-specific Abs deliver conjugated Ags to DCs with high specificity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efficiency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efficiencies. Thus, we found no evidence of CPP specifically favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the specificity with which Abs recognize their targets, this favors the use of DC receptor-specific Abs for in vivo vaccination strategies.
B Cell Depletion Inhibits Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice
Authors: Yu Shiguang , Dunn Robert , Kehry Marilyn R. , Braley-Mullen Helen
Published: April 2014

B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50–80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.
The Role of Rearrangement at the Second Ig Heavy Chain Locus in Maintaining B Cell Tolerance to DNA
Authors: Liu Yang , Li Lisa , Mohan Chandra
Published: April 2014

In recently generated B6.56R anti-DNA autoantibody-transgenic mice, it was noted that a substantial fraction of the B cells that had avoided DNA reactivity had done so through the rearrangement and usage of the endogenous, nontargeted H chain (HC) allele. This suggested that rearrangement at the second HC locus might be an important mechanism through which self-reactive B cells might successfully revise their initial Ag specificity. To test the importance of this mechanism in B cell tolerance, we generated B6.56R/56R mice that possessed the 56R anti-DNA H chain transgene inserted into both HC loci. These transgenic homozygotes developed higher titers of anti-DNA Abs, with an expanded population of B220lowMHC class IIlow B cells, enriched for CD21lowCD23low preplasmablasts. The analysis of hybridomas from these mice revealed that the only avenue by which these B cells could avoid DNA reactivity was through the use of the editor L chains, Vk20 or Vk21. Hence, in addition to LC editing, rearrangement and usage of the second HC locus/allele constitutes an important safety valve for B cells the primary BCR of which confers DNA reactivity. In contrast to these tolerance mechanisms, editing the first rearranged HC locus (through HC replacement) and somatic mutations appear to be less frequently used to edit/revise self-reactive B cells.
Presence of Suppressor HIV-Specific CD8+ T Cells Is Associated with Increased PD-1 Expression on Effector CD8+ T Cells
Authors: Elrefaei Mohamed , Baker Chris A. R. , Jones Norman G. , Bangsberg David R. , Cao Huyen
Published: April 2014

Mechanisms leading to the observed immune dysregulation in HIV-1 infection are not well understood. HIV-specific IL-10-positive CD8+ T cells are increased in advanced HIV disease. We have previously reported that Gag-specific IL-10-positive CD8+ T cells suppressed cytolysis. In this study we describe the suppressive effect of Nef-specific IL-10-positive CD8+ T cells. Interestingly, simultaneous removal of both Gag- and Nef-specific IL-10-positive CD8+ T cells led to higher HIV-specific cytolysis compared with the removal of Nef-specific IL-10-positive CD8+ T cells alone. We also examined the level of programmed cell death-1 (PD-1) as a measure of immune dysfunction in association with IL-10-positive suppressor CD8+ T cells. The level of PD-1 expression on CD107-positive effector CD8+ T cells was significantly increased when IL-10-positive suppressor CD8+ T cells were present (p < 0.05). Our results suggest that IL-10-positive suppressor CD8+ T cells contribute to the immune dysfunction observed in advanced HIV infection and that the concomitant presence of multiple IL-10-positive CD8+ T cell populations may have an additive suppressive effect.
The Innate NK Cells, Allograft Rejection, and a Key Role for IL-15
Authors: Kroemer Alexander , Xiao Xiang , Degauque Nicolas , Edtinger Karoline , Wei Haiming , Demirci Gulcin , Li Xian Chang
Published: April 2014

Transplant rejection is mediated primarily by adaptive immune cells such as T cells and B cells. The T and B cells are also responsible for the specificity and memory of the rejection response. However, destruction of allografts involves many other cell types including cells in the innate immune system. As the innate immune cells do not express germline-encoded cell surface receptors that directly recognize foreign Ags, these cells are thought to be recruited by T cells to participate in the rejection response. In this study, we examined the alloreactivity of the innate NK cells in Rag−/− mice using a stringent skin transplant model and found that NK cells at a resting state readily reject allogeneic cells, but not the skin allografts. We also found that IL-15, when preconjugated to its high affinity IL-15Rα-chain, is remarkably potent in stimulating NK cells in vivo, and NK cells stimulated by IL-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells. Furthermore, NK cell-mediated graft rejection does not show features of memory responses. Our data demonstrate that NK cells are potent alloreactive cells when fully activated and differentiated under certain conditions. This finding may have important clinical implications in models of transplantation and autoimmunity.
Comprehensive Gene Expression Profiling of Peyer’s Patch M Cells, Villous M-Like Cells, and Intestinal Epithelial Cells
Authors: Terahara Kazutaka , Yoshida Masato , Igarashi Osamu , Nochi Tomonori , Pontes Gemilson Soares , Hase Koji , Ohno Hiroshi , Kurokawa Shiho , Mejima Mio , Takayama Naoko , Yuki Yoshikazu , Lowe Anson W. , Kiyono Hiroshi
Published: April 2014

Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer’s patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.
Myeloid-Derived Suppressor Cells Accumulate in Kidney Allograft Tolerance and Specifically Suppress Effector T Cell Expansion
Authors: Dugast Anne-Sophie , Haudebourg Thomas , Coulon Flora , Heslan Michèle , Haspot Fabienne , Poirier Nicolas , Vuillefroy de Silly Romain , Usal Claire , Smit Helga , Martinet Bernard , Thebault Pamela , Renaudin Karine , Vanhove Bernard
Published: April 2014

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.
Central Memory CD8+ T Cells Appear to Have a Shorter Lifespan and Reduced Abundance as a Function of HIV Disease Progression
Authors: Ladell Kristin , Hellerstein Marc K. , Cesar Denise , Busch Robert , Boban Drina , McCune Joseph M.
Published: April 2014

Progressive HIV disease has been associated with loss of memory T cell responses to Ag. To better characterize and quantify long-lived memory T cells in vivo, we have refined an in vivo labeling technique to study the kinetics of phenotypically distinct, low-frequency CD8+ T cell subpopulations in humans. HIV-negative subjects and antiretroviral-untreated HIV-infected subjects in varying stages of HIV disease were studied. After labeling the DNA of dividing cells with deuterated water (2H2O), 2H-label incorporation and die-away kinetics were quantified using a highly sensitive FACS/mass spectrometric method. Two different populations of long-lived memory CD8+ T cells were identified in HIV-negative subjects: CD8+CD45RA−CCR7+CD28+ central memory (TCM) cells expressing IL-7Rα and CD8+CD45RA+CCR7−CD28− RA effector memory (TEMRA) cells expressing CD57. In pilot studies in HIV-infected subjects, TCM cells appeared to have a shorter half-life and reduced abundance, particularly in those with high viral loads; TEMRA cells, by contrast, retained a long half-life and accumulated in the face of progressive HIV disease. These data are consistent with the hypothesis that IL-7Rα+ TCM cells represent true memory CD8+ T cells, the loss of which may be responsible in part for the progressive loss of T cell memory function during progressive HIV infection.
Chronic Estradiol Administration In Vivo Promotes the Proinflammatory Response of Macrophages to TLR4 Activation: Involvement of the Phosphatidylinositol 3-Kinase Pathway
Authors: Calippe Bertrand , Douin-Echinard Victorine , Laffargue Muriel , Laurell Henrik , Rana-Poussine Vanessa , Pipy Bernard , Guéry Jean-Charles , Bayard Francis , Arnal Jean-François , Gourdy Pierre
Published: April 2014

Short-term exposure to 17β-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor α (ERα)-dependent activation of the PI3K pathway. In the present study, we confirm that in vitro exposure of mouse peritoneal macrophages to E2 enhanced Akt phosphorylation and slightly decreased LPS-induced cytokine production. In striking contrast, we show that chronic administration of E2 to ovariectomized mice markedly increases the expression of IL-1β, IL-6, IL-12p40, and inducible NO synthase by resident peritoneal macrophages in response to LPS ex vivo. These results clearly indicate that short-term E2 treatment in vitro does not predict the long-term effect of estrogens in vivo on peritoneal macrophage functions. We show that this in vivo proinflammatory effect of E2 was mediated through ERα. Although the expression of components of the LPS-recognition complex remained unchanged, we provided evidences for alterations of the TLR4 signaling pathway in macrophages from E2-treated mice. Indeed, E2 treatment resulted in the inhibition of PI3K activity and Akt phosphorylation in LPS-activated macrophages, whereas NF-κB p65 transcriptional activity was concomitantly increased. Incubation of macrophages with the PI3K inhibitor wortmanin enhanced proinflammatory cytokine gene expression in response to TLR4 activation, and abolishes the difference between cells from placebo- or E2-treated mice, demonstrating the pivotal role of the PI3K/Akt pathway. We conclude that the macrophage activation status is enhanced in vivo by E2 through ERα and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.
T-Bet Plays a Key Role in NK-Mediated Control of Melanoma Metastatic Disease
Authors: Werneck Miriam B. F. , Lugo-Villarino Geanncarlo , Hwang Eun Sook , Cantor Harvey , Glimcher Laurie H.
Published: April 2014

Antitumor responses depend on type 1 immunity, which is severely impaired in mice deficient for the T-box expressed in T cells (T-bet) transcription factor. Both T-bet-deficient (T-bet−/−) NK and CTL show defective function, which can be overcome by strong stimuli due to the expression of eomesodermin, another member of the T-box family. The effective response from T-bet−/− mice to viral infection and tumor initiation corroborates with these findings. However, T-bet−/− animals fail to control cancer metastasis and are, therefore, highly susceptible to tumor spread. The mechanism of T-bet-dependent resistance to metastatic disease is not known. In this study, we show that T-bet plays a role in inhibiting cancer metastasis by regulating the longevity and function of NK cells. Our data demonstrate that the absence of a proper innate immune response driven by NK cells in T-bet−/− mice precludes the initiation of a potent adaptive response to tumors. Adoptive transfer of wild-type activated NK cells protects T-bet−/− animals after melanoma challenge showing that reconstitution of the NK compartment in these mice is sufficient to mediate a significant reduction in tumor burden. Transfer of T-bet−/− A-NK cells fails to do so, due to their reduced in vivo survival, inefficient lysis of cancer cells, and poor IFN-γ production. Taken together, these results show for the first time an irreplaceable role for T-bet in the NK-mediated cross-talk between innate and adaptive immune responses to metastatic disease.
Blunted IgE-Mediated Activation of Mast Cells in Mice Lacking the Ca2+-Activated K+ Channel KCa3.1
Authors: Shumilina Ekaterina , Lam Rebecca S. , Wölbing Florian , Matzner Nicole , Zemtsova Irina M. , Sobiesiak Malgorzata , Mahmud Hasan , Sausbier Ulrike , Biedermann Tilo , Ruth Peter , Sausbier Matthias , Lang Florian
Published: April 2014

Mast cell stimulation by Ag is followed by the opening of Ca2+-activated K+ channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca2+-activated K+ channel KCa3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of KCa3.1 knockout mice (KCa3.1−/−) and their wild-type littermates (KCa3.1+/+). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcεRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in KCa3.1−/− BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca2+ ionophore ionomycin was followed by stimulation of Ca2+-activated K+ channels and cell membrane hyperpolarization in KCa3.1+/+, but not in KCa3.1−/− BMMCs. Upon Ag stimulation, Ca2+ entry but not Ca2+ release from intracellular stores was markedly impaired in KCa3.1−/− BMMCs. Similarly, Ca2+ entry upon endothelin-1 stimulation was significantly reduced in KCa3.1−/− cells. Ag-induced release of β-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in KCa3.1−/− BMMCs compared with KCa3.1+/+ BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in KCa3.1−/− cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by ∼50%) blunted in KCa3.1−/− mice. In conclusion, KCa3.1 is required for Ca2+-activated K+ channel activity and Ca2+-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.
Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival
Authors: Melcher Martin , Unger Bernd , Schmidt Uwe , Rajantie Iiro A. , Alitalo Kari , Ellmeier Wilfried
Published: April 2014

Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec−/−Btk−/− BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec−/−Btk−/− macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor α (GM-CSFRα) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage-specific regulation of GM-CSFRα expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling-induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM-CSFRα expression.
Host T Cells Are the Main Producers of IL-17 within the Central Nervous System during Initiation of Experimental Autoimmune Encephalomyelitis Induced by Adoptive Transfer of Th1 Cell Lines
Authors: Lees Jason R. , Iwakura Yoichiro , Russell John H.
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4+ T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both αβ and γδ TCR+ T cells with a CD4+CD8− or CD4−CD8− phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.
CD47 Expression on T Cell Is a Self-Control Negative Regulator of Type 1 Immune Response
Authors: Bouguermouh Salim , Van Vu Quang , Martel Julie , Gautier Patrick , Rubio Manuel , Sarfati Marika
Published: April 2014

The cytokine milieu and dendritic cells (DCs) direct Th1 development. Yet, the control of Th1 polarization by T cell surface molecules remains ill-defined. We here report that CD47 expression on T cells serves as a self-control mechanism to negatively regulate type 1 cellular and humoral immune responses in vivo. Th2-prone BALB/c mice that lack CD47 (CD47−/−) displayed a Th1-biased Ab profile at steady state and after immunization with soluble Ag. CD47−/− mice mounted a T cell-mediated exacerbated and sustained contact hypersensitivity (CHS) response. After their adoptive transfer to naive CD47-deficient hosts 1 day before immunization with soluble Ag, CD47−/− as compared with CD47+/+CD4+ transgenic (Tg) T cells promoted the deviation of Ag-specific T cell responses toward Th1 that were characterized by a high IFN-γ:IL-4 cytokine ratio. Although selective CD47 deficiency on DCs led to increased IL-12p70 production, CD47−/−Tg T cells produced more IFN-γ and displayed higher T-bet expression than CD47+/+ Tg T cells in response to OVA-loaded CD47−/− DCs. CD47 as part of the host environment has no major contribution to the Th1 polarization responses. We thus identify the CD47 molecule as a T cell-negative regulator of type 1 responses that may limit unwanted collateral damage to maximize protection and minimize host injury.
Full Development of Th2 Immunity Requires Both Innate and Adaptive Sources of CD154
Authors: Jenkins Stephen J. , Perona-Wright Georgia , MacDonald Andrew S.
Published: April 2014

The CD40-CD154 interaction is critical for Th2 response generation during helminth infection and following immunization with helminth-conditioned dendritic cells, yet the key cellular sources of these molecules have still to be defined in vivo. In this study, we demonstrate that the requirement for CD40 expression during murine Th2 response induction is restricted exclusively to the Ag-bearing dendritic cells. In contrast, development of full Th2 immunity required CD154 expression on multiple populations. In this respect, optimal production of IL-5, IL-10, and IL-13 was dependent upon CD154 expression by both CD4+ T cells and non-lymphoid cells. IL-4 production had less stringent costimulatory requirements, with expression of CD154 on either non-lymphoid cells or T cells alone being sufficient to enable production of this archetypal Th2 cytokine. Disparities in CD154 requirements for T cell and B cell responses were revealed during experimental schistosomiasis where, even in the face of robust Th2 generation, B cell class-switching was entirely dependent upon expression of CD154 by the lymphoid compartment. These data help define the costimulatory interactions that occur during the generation of Th2 immunity, and challenge the widely held view that CD154 expressing T cells are the sole contributors in this process.
Induction of Exosome Release in Primary B Cells Stimulated via CD40 and the IL-4 Receptor
Authors: Saunderson Sarah C. , Schuberth Petra C. , Dunn Amy C. , Miller Lilija , Hock Barry D. , MacKay Philippa A. , Koch Norbert , Jack Ralph W. , McLellan Alexander D.
Published: April 2014

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed, marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not R constitutive activity of B cells, but may be induced following cell: cell signaling.
Cell Intrinsic TGF-β1 Regulation of B Cells
Authors: Gros Marilyn J. , Naquet Philippe , Guinamard Rodolphe R.
Published: April 2014

TGF-β family cytokines play multiple roles in immune responses. TGF-β1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-β1 phenotype, as deficiency of TGF-β1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-β1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-β1 control of B cells. The analysis of T cell-deficient, TGF-β1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-β1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-β1.
Molecular Basis of the Dual Functions of 2B4 (CD244)
Authors: Chlewicki Lukasz K. , Velikovsky C. Alejandro , Balakrishnan Vamsi , Mariuzza Roy A. , Kumar Vinay
Published: April 2014

2B4 belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+ (αβ) T cells. The murine NK receptor 2B4 exhibits both inhibitory and activating functions, whereas human 2B4 has been reported to be an activating molecule. How murine 2B4 can act both as an activating and inhibitory receptor and what distinguishes its function from human 2B4 have remained largely unknown. We use here a model system that allows the study of human and murine 2B4 under identical and controlled conditions. These studies reveal that both human and mouse 2B4 can activate or inhibit NK cells. We show here that the level of 2B4 expression and the degree of 2B4 cross-linking play a significant role in the regulation of signaling lymphocyte activation molecule-associated protein-mediated activation by 2B4. A high level of 2B4 expression, heavy cross-linking, and relative paucity of signaling lymphocyte activation molecule-associated protein promote inhibitory function. Our studies demonstrate how a single receptor can have opposing function depending on the degree of receptor expression, extent of its ligation, and the relative abundance of certain adaptor molecules. Because the levels of 2B4 and CD48 are dynamically regulated, these findings have implications for the regulation of NK cell function.
NK Cell Protease Granzyme M Targets α-Tubulin and Disorganizes the Microtubule Network
Authors: Bovenschen Niels , de Koning Pieter J. A. , Quadir Razi , Broekhuizen Roel , Damen J. Mirjam A. , Froelich Christopher J. , Slijper Monique , Kummer J. Alain
Published: April 2014

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component α-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, α-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct α-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.
Platelet-Activating Factor-Mediated Endosome Formation Causes Membrane Translocation of p67phox and p40phox That Requires Recruitment and Activation of p38 MAPK, Rab5a, and Phosphatidylinositol 3-Kinase in Human Neutrophils
Authors: McLaughlin Nathan J. D. , Banerjee Anirban , Khan Samina Y. , Lieber Janet L. , Kelher Marguerite R. , Gamboni-Robertson Fabia , Sheppard Forest R. , Moore Ernest E. , Mierau Gary W. , Elzi David J. , Silliman Christopher C.
Published: April 2014

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 μM PAF for 1–3 min, and in some cases activated with PMA, and O2− was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy. PAF caused activation of dynamin-2, resulting in endosomal formation that required PI3K and contained early endosomal Ag-1 (EEA-1) and Rab5a. The apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK signalosome assembled on Rab5a and phosphorylated EEA-1 and Rab GDP dissociation inhibitor, with the latter causing Rab5a activation. Electron microscopy demonstrated that PAF caused two distinct sites for activation of p38 MAPK. EEA-1 provided a scaffold for recruitment of the p40phox-p67phox complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40phox-p67phox localizing to gp91phox, which was PI3K-, but not p47phox-, dependent. In conclusion, PAF transduces signals through CME, and such GPCR signaling may allow for pharmacological manipulation of these cells to decrease PMN-mediated acute organ injury.
Two Different Transcription Factors Discriminate the −315C>T Polymorphism of theFcεRIα Gene: Binding of Sp1 to −315C and of a High Mobility Group-Related Molecule to −315T
Authors: Kanada Shunsuke , Nakano Nobuhiro , Potaczek Daniel P. , Maeda Keiko , Shimokawa Naomi , Niwa Yusuke , Fukai Tatsuo , Sanak Marek , Szczeklik Andrew , Yagita Hideo , Okumura Ko , Ogawa Hideoki , Nishiyama Chiharu
Published: April 2014

The α-chain is a specific component of FcεRI, which is essential for the cell surface expression of FcεRI and the binding of IgE. Recently, two single nucleotide polymorphisms (SNPs) in the α-chain promoter, −315C>T and −66T>C, have been shown by statistic studies to associate with allergic diseases. The effect of −66 SNP on GATA-1-mediated promoter activity has been already indicated. In the present study, to investigate roles of the −315 SNP on the α-chain promoter functions, the transcription activity was evaluated by reporter assay. The α-chain promoter carrying −315T (minor allele) possessed significantly higher transcriptional activity than that of −315C (major allele). EMSA indicated that the transcription factor Sp1, but not Myc-associated zinc finger protein (MAZ), was bound to the −315C allele probe and that a transcription factor belonging to a high mobility group-family bound to the −315T allele probe. The chromatin immunoprecipitation assay suggested that high mobility group 1, 2, and Sp1 bound around −315 of FcεRIα genomic DNA in vivo in the human basophil cell line KU812 with −315C/T and in human peripheral blood basophils with −315C/C, respectively. When cell surface expression level of FcεRI on basophils was analyzed by flow cytometry, basophils from individuals carrying −315T allele expressed significantly higher amount of FcεRI compared with those of −315C/C. The findings demonstrate that a −315 SNP significantly affects human FcεRI α-chain promoter activity and expression level of FcεRI on basophils by binding different transcription factors to the SNP site.
Immunotherapy with CpG Oligonucleotides and Antibodies to TNF-α Rescues Neonatal Mice from Lethal Arenavirus-Induced Meningoencephalitis
Authors: Pedras-Vasconcelos João A. , Puig Montserrat , Sauder Christian , Wolbert Candie , Ovanesov Mikhail , Goucher David , Verthelyi Daniela
Published: April 2014

Viral encephalitides are life-threatening diseases in neonates partly due to the irreversible damage inflammation causes to the CNS. This study explored the role of proinflammatory cytokines in the balance between controlling viral replication and eliciting pathologic immune responses in nonlytic viral encephalitis. We show that neonatal mice challenged with arenavirus Tacaribe (TCRV) develop a meningoencephalitis characterized by high IFN-γ and TNF-α levels and mild T cell infiltration. Neutralization of the TNF-α using mAb was associated with lower chemokine expression, reduced T cell infiltration, and lower levels of IFN-γ, and TNF-α in the CNS and led to 100% survival. Moreover, treatment with Abs to TNF-α improved mobility and increased survival even after the mice developed bilateral hind limb paralysis. Of note, animals treated with anti-TNF-α Abs alone did not clear the virus despite generating Abs to TCRV. Direct activation of the innate immune response using CpG oligodeoxynucleotides in combination with anti-TNF-α Abs resulted in 100% survival and complete viral clearance. To our knowledge, this is the first demonstration of the use of innate immune modulators plus Abs to TNF-α as therapeutics for a lethal neurotropic viral infection.
Francisella Targets Cholesterol-Rich Host Cell Membrane Domains for Entry into Macrophages
Authors: Tamilselvam Batcha , Daefler Simon
Published: April 2014

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains (“lipid rafts”) with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.
E1A Oncogene Enhancement of Caspase-2-Mediated Mitochondrial Injury Sensitizes Cells to Macrophage Nitric Oxide-Induced Apoptosis
Authors: Radke Jay R. , Siddiqui Zeba K. , Miura Tanya A. , Routes John M. , Cook James L.
Published: April 2014

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF α and NO. E1A sensitizes cells to TNF by repressing its activation of NF-κB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-κB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-κB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-κB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.
Flagellin Treatment Protects against Chemicals, Bacteria, Viruses, and Radiation
Authors: Vijay-Kumar Matam , Aitken Jesse D. , Sanders Catherine J. , Frias Amena , Sloane Valerie M. , Xu Jianguo , Neish Andrew S. , Rojas Mauricio , Gewirtz Andrew T.
Published: April 2014

Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. A potential means of rapidly protecting such populations might be to activate innate host defense pathways, which can provide broad protection against a variety of insults. However, innate immune activators can, by themselves, result in severe inflammatory pathology, which in large part is driven by hemopoietic-derived cytokines such as TNF-α. We reasoned that, because it preferentially activates epithelial cells, the TLR5 agonist flagellin might not induce severe inflammatory pathology and yet be an ideal agent to provide such non-specific protection, particularly at the mucosal surfaces that serve as a front line of host defense. In accordance, we observed that systemic treatment of mice with purified flagellin did not induce the serologic, histopathologic, and clinical hallmarks of inflammation that are induced by LPS but yet protected mice against chemicals, pathogens, and ionizing radiation. Flagellin-elicited radioprotection required TLR5, the TLR signaling adaptor MyD88, and was effective if given between 2 h before to 4 h after exposure to irradiation. Flagellin-elicited radioprotection was, in part, mediated via effects on cells in bone marrow but yet rescued mortality without a pronounced rescue of radiation-induced anemia or leukopenia. Thus, systemic administration of flagellin may be a relatively safe means of providing temporary non-specific protection against a variety of challenges.