bluuebunny/crossref_metadata_2025 huggingface dataset:
CD16+ Monocyte-Derived Macrophages Activate Resting T Cells for HIV Infection by Producing CCR3 and CCR4 Ligands
Authors: Ancuta Petronela , Autissier Patrick , Wurcel Alysse , Zaman Tauheed , Stone David , Gabuzda Dana
Published: April 2014

The CD16+ monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16+ and CD16− Mo subsets to render resting CD4+ T cells permissive for HIV replication. We demonstrate that CD16+ Mo preferentially differentiate into macrophages (Mφ) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16+, but not CD16−, Mo-derived Mφ from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16− Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3+ and CCR4+ T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16+ Mo/Mφ. Thus, expansion of CD16+ Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.
Deficit of CD47 Results in a Defect of Marginal Zone Dendritic Cells, Blunted Immune Response to Particulate Antigen and Impairment of Skin Dendritic Cell Migration
Authors: Hagnerud Sven , Manna Partha Pratim , Cella Marina , Stenberg Åsa , Frazier William A. , Colonna Marco , Oldenborg Per-Arne
Published: April 2014

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein α, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47−/− mice exhibit a selective reduction of splenic CD11chighCD11bhighCD8α−CD4+ DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein α, possibly explaining their selective deficiency in CD47−/− mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47−/− mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47−/− mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.
Highly Oligomeric Procyanidins Ameliorate Experimental Autoimmune Encephalomyelitis via Suppression of Th1 Immunity
Authors: Miyake Mika , Sasaki Katsunori , Ide Kazuki , Matsukura Yasuko , Shijima Kumiko , Fujiwara Daisuke
Published: April 2014

Extracts of Jatoba, a South American herb, when injected i.p. into a mouse model of experimental autoimmune encephalomyelitis (EAE), inhibited the aggravation of clinical symptoms. At the same time, production of myelin oligodendrocyte glycoprotein Ag-specific IFN-γ and TNF-α by spleen cells was markedly suppressed. After administration of Jatoba there was minimal evidence of the demyelination that is characteristic of the EAE model. Decreases in clinical scores were observed when Jatoba extracts were injected just before Ag. The purified active compounds are likely to be polyphenols that are absorbable to polyvinylpolypyrrolidone. The active compounds were polymerized polyphenol polymers (procyanidins) and at least five degrees of polymerization were necessary for activity. In addition, extracts of other plant materials containing such procyanidins had similar activity. After administration of highly polymerized procyanidins, there was a decrease in both dendritic and CD4+ T cells. Although macrophages were increased in number, the expression of CD80 and MHC class II molecules was depressed indicating that the macrophages were immature. The results indicate that the suppression of development of EAE by the highly polymerized procyanidins resulted from an inhibition of Th1 and the effects might be associated with depression of Ag-presenting capability.
Th2 Cytokines Down-Regulate TLR Expression and Function in Human Intestinal Epithelial Cells
Authors: Mueller Tobias , Terada Tomohiro , Rosenberg Ian M. , Shibolet Oren , Podolsky Daniel K.
Published: April 2014

TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1 cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13) on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR function was determined by I-κBα phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the requisite TLR4 coreceptor MD-2. TLR4/MD-2-mediated LPS uptake and TLR ligand-induced I-κBα phosphorylation and IL-8 secretion were significantly diminished in Th2 cytokine-primed IECs. The down-regulatory effect of Th2 cytokines on TLR expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine IFN-γ. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling, but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing TLR signaling in human IECs.
Novel Function of Alternatively Activated Macrophages: Stabilin-1-Mediated Clearance of SPARC
Authors: Kzhyshkowska Julia , Workman Gail , Cardó-Vila Marina , Arap Wadih , Pasqualini Renata , Gratchev Alexei , Krusell Liis , Goerdt Sergij , Sage E. Helene
Published: April 2014

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.
CD64-Directed Immunotoxin Inhibits Arthritis in a Novel CD64 Transgenic Rat Model
Authors: van Vuuren Anneke J. , van Roon Joel A. G. , Walraven Vanessa , Stuij Ilonka , Harmsen Martin C. , McLaughlin Pamela M. J. , van de Winkel Jan G. J. , Thepen Theo
Published: April 2014

Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages have increased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin, composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we show elevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directed immunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing macrophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established an adjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulation in this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results in significant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophage numbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data support synovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination of inflammatory macrophages through a CD64-directed immunotoxin may provide a novel approach for treatment of RA.
TLR4 Signaling Attenuates Ongoing Allergic Inflammation
Authors: Hollingsworth John W. , Whitehead Gregory S. , Lin Kaifeng Lisa , Nakano Hideki , Gunn Michael D. , Schwartz David A. , Cook Donald N.
Published: April 2014

The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure.
Increased Expression of Ifi202, an IFN-Activatable Gene, in B6.Nba2 Lupus Susceptible Mice Inhibits p53-Mediated Apoptosis
Authors: Xin Hong , D’Souza Sanjay , Jørgensen Trine N. , Vaughan Andrew T. , Lengyel Peter , Kotzin Brian L. , Choubey Divaker
Published: April 2014

Increased expression of p202 protein (encoded by the Ifi202 gene) in splenocytes derived from B6.Nba2 mice (congenic for the Nba2 interval derived from the New Zealand Black mice) was correlated with defects in apoptosis of splenic B cells and increased susceptibility to develop systemic lupus erythematosus. We have now investigated the molecular mechanisms by which increased expression of p202 in B6.Nba2 cells contributes to defects in apoptosis. In this study, we report that increased expression of p202 in the B6.Nba2 splenocytes, as compared with cells derived from the parental C57BL/6 (B6) mice, was correlated with increased levels of p53 protein and inhibition of p53-mediated transcription of target genes that encode proapoptotic proteins. Conversely, knockdown of p202 expression in B6.Nba2 cells resulted in stimulation of p53-mediated transcription. We found that p202 bound to p53 in the N-terminal region (aa 44–83) comprising the proline-rich region that is important for p53-mediated apoptosis. Consistent with the binding of p202 to p53, increased expression of p202 in B6.Nba2 mouse embryonic fibroblasts inhibited UV-induced apoptosis. Taken together, our observations support the idea that increased expression of p202 in B6.Nba2 mice increases the susceptibility to develop lupus, in part, by inhibiting p53-mediated apoptosis.
Expression of TCRαβ Partly Rescues Developmental Arrest and Apoptosis of αβ T cells in Bcl11b−/− Mice
Authors: Inoue Jun , Kanefuji Tsutomu , Okazuka Kiyoshi , Watanabe Hisami , Mishima Yukio , Kominami Ryo
Published: April 2014

Bcl11b −/− mice show developmental arrest at the CD44−CD25+ double-negative 3 (DN3) or immature CD8+single-positive stage of αβ T cell. We have performed detailed analysis of sorted subsets of Bcl11b−/− thymocytes, DN3 and CD44−CD25− double-negative 4 (DN4) cells. Surface expression of TCRβ proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRβ proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRβ or TCRαβ expression on the cell surface failed to promote transition from the DN3 to CD4+CD8+ double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b−/− DN3 thymocytes showed normal DNA rearrangements between Dβ and Jβ segments but limited DNA rearrangements between Vβ and DJβ without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b−/− DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vβ and Dβ gene locus. Analysis of Bcl11b−/− DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-xL and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRαβ in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.
Arginine Transport via Cationic Amino Acid Transporter 2 Plays a Critical Regulatory Role in Classical or Alternative Activation of Macrophages
Authors: Yeramian Andrée , Martin Lorena , Serrat Neus , Arpa Luis , Soler Concepció , Bertran Joan , McLeod Carol , Palacín Manuel , Modolell Manuel , Lloberas Jorge , Celada Antonio
Published: April 2014

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases l-arginine transport only through the y+ system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in l-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.
Targeting Melanoma Cells with Human High Molecular Weight-Melanoma Associated Antigen-Specific Antibodies Elicited by a Peptide Mimotope: Functional Effects
Authors: Luo Wei , Ko Eric , Hsu Jeff Chi-feng , Wang Xinhui , Ferrone Soldano
Published: April 2014

Human high molecular weight-melanoma associated Ag (HMW-MAA) mimics have been shown to elicit HMW-MAA-specific humoral immune responses that appear to be clinically beneficial. This finding has stimulated interest in characterizing the mechanism(s) underlying the ability of the elicited Abs to exert an anti-tumor effect. To address this question, in the present study, we have generated HMW-MAA-specific Abs by sequentially immunizing rabbits with the peptide P763.74, which mimics the HMW-MAA determinant recognized by mAb 763.74, and with HMW-MAA+ melanoma cells. HMW-MAA-specific Abs isolated from immunized rabbits mediated cell-dependent cytotoxicity but did not mediate complement-dependent cytotoxicity of HMW-MAA+ melanoma cells. These Abs also effectively inhibited spreading, migration and Matrigel invasion of HMW-MAA+ melanoma cells. Besides contributing to our understanding of the role of HMW-MAA in the biology of melanoma cells, these results suggest that both immunological and nonimmunological mechanisms underlie the beneficial clinical effects associated with the induction of HMW-MAA-specific Abs in melanoma patients immunized with a HMW-MAA mimic.
The Proteasome Pathway Destabilizes Yersinia Outer Protein E and Represses Its Antihost Cell Activities
Authors: Ruckdeschel Klaus , Pfaffinger Gudrun , Trülzsch Konrad , Zenner Gerhardt , Richter Kathleen , Heesemann Jürgen , Aepfelbacher Martin
Published: April 2014

Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.
Defective Adhesion in Tumor Infiltrating CD8+ T Cells
Authors: Koneru Mythili , Monu Ngozi , Schaer David , Barletta Justine , Frey Alan B.
Published: April 2014

CD8+ tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56lck activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells.
Roles of the Alternative Complement Pathway and C1q during Innate Immunity to Streptococcus pyogenes
Authors: Yuste Jose , Ali Sifot , Sriskandan Shiranee , Hyams Catherine , Botto Marina , Brown Jeremy S.
Published: April 2014

Complement is important for innate immunity to the common bacterial pathogen Streptococcus pyogenes, but the relative importance of the alternative and classical pathways has not been investigated. Using mice and human serum deficient in either C1q, the first component of the classical pathway, or factor B, an important component of the alternative pathway, we have investigated the role of both pathways for innate immunity to S. pyogenes. C3b deposition on four different strains of S. pyogenes was mainly dependent on factor B. As a consequence opsonophagocytosis of S. pyogenes was reduced in serum from factor B-deficient mice, and these mice were very susceptible to S. pyogenes infection. In contrast, C3b deposition was not dependent on C1q for two of the strains investigated, H372 and H305, yet opsonophagocytosis of all four S. pyogenes strains was impaired in serum deficient in C1q. Furthermore, infection in C1q-deficient mice with strain H372 resulted in a rapidly progressive disease associated with large numbers of bacteria in target organs. These results demonstrate the important role of the alternative pathway and C1q for innate immunity to S. pyogenes and suggest that C1q-mediated innate immunity to at least some strains of S. pyogenes may involve mechanisms that are independent of C3b on the bacteria.
CD94/NKG2A Expression Is Associated with Proliferative Potential of CD8 T Cells during Persistent Polyoma Virus Infection
Authors: Byers Anthony M. , Andrews Nicolas P. , Lukacher Aron E.
Published: April 2014

Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A+ polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A− polyoma-specific CD8 T cells. Importantly, CD94/NKG2A+ anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A+ subpopulation represents a less differentiated state than the CD94/NKG2A− subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections.
MyD88-Dependent IFN-γ Production by NK Cells Is Key for Control ofLegionella pneumophilaInfection
Authors: Spörri Roman , Joller Nicole , Albers Urs , Hilbi Hubert , Oxenius Annette
Published: April 2014

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium in aquatic systems and an opportunistic intracellular pathogen in immunocompromised humans causing a severe pneumonia known as Legionnaires’ disease. Using a mouse model, we investigated molecular and cellular players in the innate immune response to infection with Lpn. We observed robust levels of inflammatory cytokines in the serum upon intranasal or i.v. infection with live, virulent Lpn, but not with inactivated or avirulent bacteria lacking the Icm/Dot type IV secretion system. Interestingly, Lpn-induced serum cytokines were readily detectable regardless of the capacity of Icm/Dot-proficient Lpn to replicate in host cells and the Lpn permissiveness of the host mice. We found NK cell-derived IFN-γ to be the key cytokine in the resolution of Lpn infection, whereas type I IFNs did not appear to play a major role in our model. Accordingly, NK cell-depleted or IFN-II-R-deficient mice carried severely increased bacterial burdens or failed to control Lpn infection, respectively. Besides the dependence of inflammatory cytokine induction on Lpn virulence, we also demonstrate a strict requirement of MyD88 for this process, suggesting the involvement of TLRs in the recognition of Lpn. However, screening of several TLR-deficient hosts did not reveal a master TLR responsible for the sensing of an Lpn infection, but provided evidence for either redundancy of individual TLRs in Lpn recognition or TLR-independent induction of inflammatory responses.
Immature Human Dendritic Cells Infected with Leishmania infantum Are Resistant to NK-Mediated Cytolysis but Are Efficiently Recognized by NKT Cells
Authors: Campos-Martín Yolanda , Colmenares María , Gozalbo-López Beatriz , López-Núñez Marta , Savage Paul B. , Martínez-Naves Eduardo
Published: April 2014

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-γ. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.
Reevaluation of P-Selectin and α4 Integrin as Targets for the Treatment of Experimental Autoimmune Encephalomyelitis
Authors: Kerfoot Steven M. , Norman M. Ursula , Lapointe Benoit M. , Bonder Claudine S. , Zbytnuik Lori , Kubes Paul
Published: April 2014

There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate α4 integrin and P-selectin as targets for therapy in murine models of multiple sclerosis–for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either α4 integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to α4 integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-α4 integrin alone.
Fas Ligand Is Responsible for CXCR3 Chemokine Induction in CD4+ T Cell-Dependent Liver Damage
Authors: Cruise Michael W. , Lukens John R. , Nguyen Aileen P. , Lassen Matthew G. , Waggoner Stephen N. , Hahn Young S.
Published: April 2014

Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4+ T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core × TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4+ T cells into the liver of the immunized core+ TCR mice but not their core− TCR littermates. Strikingly, the inflammatory process in the liver of core+ TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-γ production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3+ lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-γ and cellular infiltration into the liver. These findings suggest that activated CD4+ T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.
Control of Metastasized Pancreatic Carcinomas in SCID/Beige Mice with Human IL-2/TKD-Activated NK Cells
Authors: Stangl Stefan , Wortmann Andreas , Guertler Ulrich , Multhoff Gabriele
Published: April 2014

Pancreatic carcinoma, the fifth leading cause of cancer-related mortality, frequently presents the stress-inducible heat shock protein 70 (Hsp70) on the cell membrane. Therefore, we explored an immunological approach exploiting the efficacy of NK cells activated either with low dose IL-2 plus Hsp70-peptide TKDNNLLGRFELSG (TKD; IL-2/TKD) or with IL-2 alone in a xenograft pancreatic carcinoma model. An orthotopic injection of either 2.5 × 106 or 1 × 106 Colo357 cells in SCID/beige mice resulted in rapidly growing primary tumors and the development of hepatic metastases on days 5 and 10, respectively. In line with results of in vitro migration assays, these NK cells also had the capacity to infiltrate pancreatic tumors and liver metastases in tumor-bearing mice. In vitro, a combined treatment of NK cells with IL-2/TKD but neither of the two treatments alone causes a profound increase in the lytic capacity against Hsp70 membrane-positive Colo357 cells. In vivo, a single i.v. injection of these NK cells on day 15 post-tumor inoculation resulted in a significant reduction in tumor weights, a delayed onset of hepatic metastases, and a prolonged life expectancy. In contrast, identically treated T cells and NK cells treated with IL-2 alone were significantly less efficient in controlling pancreatic tumors and metastases. Most importantly, four repeated i.v. infusions of IL-2/TKD-activated NK cells eradicated primary tumors and prevented hepatic metastases. In summary, our mouse data have implicated that NK cells preactivated with IL-2/TKD might provide a novel therapeutic tool for the treatment of aggressive, Hsp70-positive pancreatic carcinoma.
TNF-α Is Necessary for Induction of Coronary Artery Inflammation and Aneurysm Formation in an Animal Model of Kawasaki Disease
Authors: Hui-Yuen Joyce S. , Duong Trang T. , Yeung Rae S. M.
Published: April 2014

Kawasaki disease is the most common cause of multisystem vasculitis in childhood. The resultant coronary artery lesions make Kawasaki disease the leading cause of acquired heart disease in children in the developed world. TNF-α is a pleiotropic inflammatory cytokine elevated during the acute phase of Kawasaki disease. In this study, we report rapid production of TNF-α in the peripheral immune system after disease induction in a murine model of Kawasaki disease. This immune response becomes site directed, with migration to the coronary arteries dependent on TNF-α-mediated events. Production of TNF-α in the heart is coincident with the presence of inflammatory infiltrate at the coronary arteries, which persists during development of aneurysms. More importantly, inflammation and elastin breakdown in the coronary vessels are completely eliminated in the absence of TNF-α effector functions. Mice treated with the TNF-α-blocking agent etanercept, as well as TNFRI knockout mice, are resistant to development of both coronary arteritis and coronary aneurysm formation. Taken together, TNF-α is necessary for the development of coronary artery lesions in an animal model of Kawasaki disease. These findings have important implications for potential new therapeutic interventions in children with Kawasaki disease.
Proteasome Inhibition to Maximize the Apoptotic Potential of Cytokine Therapy for Murine Neuroblastoma Tumors
Authors: Khan Tahira , Stauffer Jimmy K. , Williams Rebecca , Hixon Julie A. , Salcedo Rosalba , Lincoln Erin , Back Timothy C. , Powell Douglas , Lockett Stephen , Arnold Alma C. , Sayers Thomas J. , Wigginton Jon M.
Published: April 2014

Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-γ plus TNF-α, we hypothesized that bortezomib also could sensitize these cells to IFN-γ plus TNF-α. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-γ and TNF-α on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-γ plus TNF-α in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-γ/TNF-α-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.
Galectin-1 Suppresses Autoimmune Retinal Disease by Promoting Concomitant Th2- and T Regulatory-Mediated Anti-Inflammatory Responses
Authors: Toscano Marta A. , Commodaro Alessandra G. , Ilarregui Juan M. , Bianco Germán A. , Liberman Ana , Serra Horacio M. , Hirabayashi Jun , Rizzo Luiz V. , Rabinovich Gabriel A.
Published: April 2014

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4+ regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.
IL-21 Enhances Tumor-Specific CTL Induction by Anti-DR5 Antibody Therapy
Authors: Smyth Mark J. , Hayakawa Yoshihiro , Cretney Erika , Zerafa Nadeen , Sivakumar Pallavur , Yagita Hideo , Takeda Kazuyoshi
Published: April 2014

Tumor cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful anti-tumor immunotherapies. In this study, we show that induction of tumor cell apoptosis by agonistic mAb against DR5, combined with delayed IL-21 treatment, suppressed tumor growth and pre-established tumor metastases. Synergistic effects of the combination were observed in several tumor models where the target tumor was sensitive to DR5-mediated apoptosis. IL-21 promoted tumor-specific CTL activity and enhanced memory responses to tumor rechallenge. These results indicate that a rational combination of Ab-based therapy that causes tumor cell apoptosis and a cytokine that promotes T cell memory is a useful new strategy for cancer immunotherapy.
Cutting Edge: Enhancement of Antibody Responses Through Direct Stimulation of B and T Cells by Type I IFN
Authors: Le Bon Agnes , Thompson Clare , Kamphuis Elisabeth , Durand Vanessa , Rossmann Cornelia , Kalinke Ulrich , Tough David F.
Published: April 2014

Type I IFN (IFN-αβ) is induced rapidly by infection and plays a key role in innate antiviral defense. IFN-αβ also exerts stimulatory effects on the adaptive immune system and has been shown to enhance Ab and T cell responses. We have investigated the importance of B and T cells as direct targets of IFN-αβ during IFN-α-mediated augmentation of the Ab response against a soluble protein Ag. Strikingly, the ability of IFN-α to stimulate the Ab response and induce isotype switching was markedly reduced in mice in which B cells were selectively deficient for the IFN-αβR. Moreover, IFN-α-mediated enhancement of the Ab response was also greatly impaired in mice in which T cells were selectively IFN-αβR-deficient. These results indicate that IFN-αβR signaling in both B and T cells plays an important role in the stimulation of Ab responses by IFN-αβ.
A Major Lung CD103 (αE)-β7 Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins
Authors: Sung Sun-Sang J. , Fu Shu Man , Rose C. Edward , Gaskin Felicia , Ju Shyr-Te , Beaty Steven R.
Published: April 2014

Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid organs. The third and largest DC population is the integrin αE (CD103) β7-positive and I-AhighCD11chigh-DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin αEβ7+ DC (αE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11bhigh myeloid and mPDCA-1+B220+Gr-1+ PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, αE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that αE-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis.
Distinct Mechanisms Control Human Naive and Antigen-Experienced CD8+ T Lymphocyte Proliferation
Authors: Migliaccio Marco , Alves Pedro Miguel Sousa , Romero Pedro , Rufer Nathalie
Published: April 2014

Human Ag-specific CD8+ T lymphocytes are heterogeneous and include functionally distinct populations. In this study, we report that at least two distinct mechanisms control the expansion of circulating naive, memory, and effector CD8+ T lymphocytes when exposed to mitogen or Ag stimulation. The first one leads to apoptosis and occurs shortly after in vitro stimulation. Susceptibility to cell death is prominent among primed T cell subsets, and it is inversely correlated with the size of the ex vivo Bcl-2high population within these subsets. Importantly, the Bcl-2high phenotype is associated to the proportion of responsive CD8+ T cells, independently of their differentiation stage. The second one depends on the expression of newly synthesized cyclin-dependent kinase inhibitor p16INK4a that occurs in a significant fraction of T cells that had been actively cycling, leading to their cell cycle arrest upon stimulation. Strikingly, accumulation of p16INK4a protein preferentially occurs in naive as opposed to primed derived T lymphocytes and is not related to apoptosis. Significant levels of p16 are readily detectable in a small number of ex vivo CD8+ T cells. Our observations reveal that activation-induced p16 expression represents an alternative process to apoptosis, limiting the proliferation potential of activated naive derived T lymphocytes.
CD8 T Cells Specific for a Donor-Derived, Self-Restricted Transplant Antigen Are Nonpathogenic Bystanders after Vascularized Heart Transplantation in Mice
Authors: Valujskikh Anna , Zhang Qiwei , Heeger Peter S.
Published: April 2014

CD8 T cell cross-priming, an established mechanism of protective antiviral immunity, was originally discovered during studies involving minor transplantation Ags. It is unclear whether or how cross-primed CD8 T cells, reactive to donor-derived, but recipient class I MHC-restricted epitopes, could injure a fully MHC-disparate, vascularized transplant. To address this question we studied host class I MHC-restricted, male transplantation Ag-reactive T cell responses in female recipients of fully MHC-disparate, male heart transplants. Cross-priming to the immune-dominant determinant HYUtyp occurred at low frequency after heart transplantation. CD8 T cell preactivation through immunization with HYUtyp mixed in CFA did not alter the kinetics of acute rejection. Furthermore, neither HYUtyp immunization nor adoptive transfer of HYUtyp-specific TCR-transgenic T cells affected outcome in 1) a model of chronic rejection in the absence of immunosuppression or 2) a model of allograft acceptance induced by costimulatory blockade. The results support the contention that CD8 T cells reactive to host-restricted, but donor-derived, Ags are highly specific and are nonpathogenic bystanders during rejection of MHC-disparate cardiac allografts.