bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17A Produced by γδ T Cells Plays a Critical Role in Innate Immunity against Listeria monocytogenes Infection in the Liver
Authors: Hamada Satoru , Umemura Masayuki , Shiono Takeru , Tanaka Kensho , Yahagi Ayano , Begum M. Dilara , Oshiro Kiyotetsu , Okamoto Yuko , Watanabe Hisami , Kawakami Kazuyoshi , Roark Christina , Born Willi K. , O'Brien Rebecca , Ikuta Koichi , Ishikawa Hiromichi , Nakae Susumu , Iwakura Yoichiro , Ohta Takao , Matsuzaki Goro
Published: April 2014

IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4+ Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR γδ T cells expressing TCR Vγ4 or Vγ6. Interestingly, TCR γδ T cells expressing both IFN-γ and IL-17A were hardly detected, indicating that the IL-17A-producing TCR γδ T cells are distinct from IFN-γ-producing γδ T cells, similar to the distinction between Th17 and Th1 in CD4+ T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR γδ T cells, which is important in innate immunity in the liver.
TLR Adaptor MyD88 Is Essential for Pathogen Control during OralToxoplasma gondiiInfection but Not Adaptive Immunity Induced by a Vaccine Strain of the Parasite
Authors: Sukhumavasi Woraporn , Egan Charlotte E. , Warren Amy L. , Taylor Gregory A. , Fox Barbara A. , Bzik David J. , Denkers Eric Y.
Published: April 2014

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88−/− mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-γ production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4+ T cell IFN-γ reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88−/− mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-γ responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.
Lipopolysaccharide Is a Direct Agonist for Platelet RNA Splicing
Authors: Shashkin Pavel N. , Brown G. Thomas , Ghosh Arundhati , Marathe Gopal K. , McIntyre Thomas M.
Published: April 2014

Platelets express TLR4 receptors, but its ligand LPS does not directly activate thrombotic functions nor, obviously, transcription by these anucleate cells. Platelets, however, store information that changes their phenotype over a few hours in the form of unprocessed RNA transcripts. We show even low concentrations of LPS in the presence of soluble CD14 initiated splicing of unprocessed IL-1β RNA, with translation and accumulation of IL-1β protein. LPS was a more robust agonist for this response than thrombin. Platelets also contained cyclooxygenase-2 pre-mRNA, which also was spliced and translated after LPS stimulation. Flow cytometry and immunocytochemistry of platelets extensively purified by negative immunodepletion showed platelets contained IL-1β, and quantitative assessment of white blood cell contamination by CD14 real time PCR confirms that leukocytes were not the IL-1β source, nor were they required for platelet stimulation. LPS did not initiate rapid platelet responses, but over time did prime platelet aggregation to soluble agonists, induced actin rearrangement, and initiated granule secretion with P-selectin expression that resulted the coating of quiescent leukocytes with activated platelets. LPS is a direct agonist for platelets that allows these cells to directly participate in the innate immune response to bacteria.
Phosphorylation of 5-Lipoxygenase at Ser523 by Protein Kinase A Determines Whether Pioglitazone and Atorvastatin Induce Proinflammatory Leukotriene B4 or Anti-Inflammatory 15-Epi-Lipoxin A4 Production
Authors: Ye Yumei , Lin Yu , Perez-Polo Jose R. , Uretsky Barry F. , Ye Zaiming , Tieu Brian C. , Birnbaum Yochai
Published: April 2014

The 5-lipoxygenase (5LO) produces leukotriene B4 and 15-epilipoxin-A4 (15-epi-LXA4). Phosphorylation at Ser523 by protein kinase A (PKA) prevents 5LO shift to the perinuclear membrane. Atorvastatin and pioglitazone up-regulate 15-epi-LXA4 production in the heart. We assessed whether phosphorylation of 5LO by PKA determines whether 5LO interacts with the membranous cytosolic phospholipase A2 (cPLA2) to produce leukotriene B4 or with cyclooxygenase-2 (COX2) to produce 15-epi-LXA4. Rats received either pioglitazone, atorvastatin, pioglitazone plus atorvastatin, vehicle, or LPS. Rat myocardial cells were incubated with pioglitazone plus atorvastatin, pioglitazone plus atorvastatin plus H-89 (PKA inhibitor), H-89, or vehicle for 8 h. Pioglitazone and atorvastatin did not affect total 5LO expression. However, both increased 5LO levels in the cytosolic fraction. H-89 caused a shift of 5LO to the membranous fraction in atorvastatin- and pioglitazone-treated rats. Pioglitazone and atorvastatin increased phospho-5LO levels. H-89 attenuated this increase. Both pioglitazone and atorvastatin increased COX2 levels in the cytosolic fraction and the membranous fraction. H-89 prevented this increase. Pioglitazone and atorvastatin increased cPLA2 expression in the membranous fraction. This effect was not attenuated by H-89. Pioglitazone plus atorvastatin increased 15-epi-LXA4 levels. H-89 attenuated the effect of pioglitazone plus atorvastatin. Pioglitazone plus atorvastatin plus H-89 increased leukotriene B4 levels. Coimmunoprecipitation showed that without H-89, atorvastatin and pioglitazone induced an interaction between 5LO and COX2 in the cytosolic fraction, whereas when H-89 was added, 5LO interacted with cPLA2 on the membranous fraction. The 5LO phosphorylation determines whether 15-epi-LXA4 (anti-inflammatory) or leukotriene B4 (inflammatory mediator) is produced.
Mitochondrial Structural Changes and Dysfunction Are Associated with Experimental Allergic Asthma
Authors: Mabalirajan Ulaganathan , Dinda Amit Kumar , Kumar Sarvesh , Roshan Reema , Gupta Pooja , Sharma Surendra Kumar , Ghosh Balaram
Published: April 2014

An imbalance between Th1 and Th2 immune response is crucial for the development of pathophysiological features of asthma. A Th2-dominant response produces oxidative stress in the airways, and it is thought to be one of the crucial components of asthma pathogenesis. Although mitochondrion is a crucial organelle to produce endogenous reactive oxygen species, its involvement in this process remains unexplored as yet. We demonstrate in this study that OVA-induced experimental allergic asthma in BALB/c mice is associated with mitochondrial dysfunction, such as reduction of cytochrome c oxidase activity in lung mitochondria, reduction in the expression of subunit III of cytochrome c oxidase in bronchial epithelium, appearance of cytochrome c in the lung cytosol, decreased lung ATP levels, reduction in the expression of 17 kDa of complex I in bronchial epithelium, and mitochondrial ultrastructural changes such as loss of cristae and swelling. However, there was no change in the expression of subunits II and III of cytochrome c oxidase. Interestingly, administration of IL-4 mAb reversed these mitochondrial dysfunction and structural changes. In contrast, IFN-γ mAb administration neither reversed nor further deteriorated the mitochondrial dysfunction and structural changes compared with control asthmatic mice administered with isotypic control Ab, although airway hyperresponsiveness deteriorated further. These results suggest that mitochondrial structural changes and dysfunction are associated with allergic asthma. These findings may help in the development of novel drug molecules targeting mitochondria for the treatment of asthma.
Tissue-Type Plasminogen Activator Is a Regulator of Monocyte Diapedesis through the Brain Endothelial Barrier
Authors: Reijerkerk Arie , Kooij Gijs , van der Pol Susanne M. A. , Leyen Thomas , van het Hof Bert , Couraud Pierre-Olivier , Vivien Denis , Dijkstra Christine D. , de Vries Helga E.
Published: April 2014

Inflammatory cell trafficking into the brain complicates several neurological disorders including multiple sclerosis. Normally, reliable brain functioning is maintained and controlled by the blood-brain barrier (BBB), which is essential to restrict the entry of potentially harmful molecules and cells from the blood into the brain. The BBB is a selective barrier formed by dedicated brain endothelial cells and dependent on the presence of intracellular tight junctions. In multiple sclerosis, a severe dysfunction of the BBB is observed, which is key to monocyte infiltration and inflammation in the brain. Proteolytic activity has been associated with these inflammatory processes in the brain. Our studies in plasma of rats indicated that the extracellular protease tissue-type plasminogen activator (tPA) correlates with the clinical signs of experimental allergic encephalomyelitis, a rat model of multiple sclerosis. In this study, we studied the function of the tPA during diapedesis of monocytes through a rat and human brain endothelial barrier. Monocyte-brain endothelial cell coculture experiments showed that monocytes induce the release of tPA by brain endothelial cells, which subsequently activates the signal transduction protein extracellular signal related kinase (ERK1/2), both involved in monocyte diapedesis. Importantly, live imaging and immunoblot analyses of rat brain endothelial cells revealed that tPA and ERK1/2 control the breakdown of the tight junction protein occludin. These studies identify tPA as a novel and relevant pathological mediator of neuroinflammation and provide a potential mechanism for this.
Transcriptional and Translational Regulation of TGF-β Production in Response to Apoptotic Cells
Authors: Xiao Yi Qun , Freire-de-Lima Celio G. , Schiemann William P. , Bratton Donna L. , Vandivier R. William , Henson Peter M.
Published: April 2014

Interaction between apoptotic cells and phagocytes through phosphatidylserine recognition structures results in the production of TGF-β, which has been shown to play pivotal roles in the anti-inflammatory and anti-immunogenic responses to apoptotic cell clearance. Using 3T3-TβRII and RAWTβRII cells in which a truncated dominant-negative TGF-β receptor II was stably transfected to avoid autofeedback induction of TGF-β, we investigate the mechanisms by which TGF-β was produced through PSRS engagement. We show, in the present study, that TGF-β was regulated at both transcriptional and translational steps. P38 MAPK, ERK, and JNK were involved in TGF-β transcription, whereas translation required activation of Rho GTPase, PI3K, Akt, and mammalian target of rapamycin with subsequent phosphorylation of translation initiation factor eukaryotic initiation factor 4E. Strikingly, these induction pathways for TGF-β production were different from those initiated in the same cells responding to LPS or PMA.
Myeloid-Related Proteins Rapidly Modulate Macrophage Nitric Oxide Production during Innate Immune Response
Authors: Pouliot Philippe , Plante Isabelle , Raquil Marie-Astrid , Tessier Philippe A. , Olivier Martin
Published: April 2014

S100A8 and S100A9 are intracellular calcium-binding proteins produced by myeloid cells that promote neutrophil/monocyte recruitment at inflamed tissues by enhancing attachment to endothelial cells. Although the intracellular functions of these proteins, i.e., myeloid-related proteins (MRP)-8 and MRP-14, are not completely understood, these proteins exhibit prominent extracellular cytokine-like functions and are considered reliable markers of inflammation in diverse diseases. As S100A8 and S100A9 have been reported to be rapidly released in response to components derived from infectious agents, we hypothesized that they play an important role in the modulation of key microbicidal phagocyte functions. In this study, we report for the first time that MRPs are powerful inducers of NO production by murine macrophages (Mφ). This increase in NO production was linked to an increased inducible NO synthase expression both at gene and protein level. This induction was concomitant with an important phosphorylation of SAPK/JNK, but also of MEK and ERK kinases. Upon stimulation with MRPs, NF-κB was rapidly translocated to the nucleus (30 min). When Mφ were treated concomitantly with IFN-γ, another activator of Mφ functions, we observed a strong synergy in NO production, synergy that resulted from the engagement of exclusive signaling pathways: SAPK/JNK, ERK and NF-κB were involved in signaling of MRPs, whereas IFN-γ uses the JAK/STAT pathway. This suggests that the synergy results from interactions of transcription factors in the promoter region. Finally, we observed this effect to be dependent on TLR4. Collectively, our study unravels the importance of MRPs as potent new inducers of Mφ NO production.
Neutrophil Apoptosis: Selective Regulation by Different Ligands of Integrin αMβ2
Authors: Pluskota Elzbieta , Soloviev Dmitry A. , Szpak Dorota , Weber Christian , Plow Edward F.
Published: April 2014

Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. αMβ2 is reported either to delay or accelerate neutrophil apoptosis, but the mechanisms by which this integrin can support such diametrically opposed responses are poorly understood. The abilities of closely related αMβ2 ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative αMβ2 recognition peptides, P1 and P2-C, differed markedly in their effects on neutrophil apoptosis. Plasminogen, fibrinogen, and P2-C suppressed apoptosis via activation of Akt and ERK1/2 kinases, while angiostatin and P1 failed to activate these prosurvival pathways and did not prevent neutrophil apoptosis. Using cells transfected with αMβ2 or its individual αM or β2 subunits, and purified receptors and its constituent chains, we show that engagement of both subunits with prosurvival ligands is essential for induction of the prosurvival response. Hence, engagement of a single integrin by closely related ligands can induce distinct signaling pathways, which can elicit distinct cellular responses.
Nucleotide Oligomerization Domains 1 and 2: Regulation of Expression and Function in Preadipocytes
Authors: Stroh Thorsten , Batra Arvind , Glauben Rainer , Fedke Inka , Erben Ulrike , Kroesen Anjo , Heimesaat Markus M. , Bereswill Stefan , Girardin Stephen , Zeitz Martin , Siegmund Britta
Published: April 2014

Translocation of bacteria into the mesenteric fat during intestinal inflammation and the expression of functional TLR1–9 in murine preadipocytes and adipocytes suggest an active role for these cells in innate immunity. The present study focuses on nucleotide oligomerization domains 1 and 2 representing intracellular pattern recognition receptors that sense motifs derived from bacterial peptidoglycans. On mRNA level nucleotide oligomerization domain 1 was found to be constitutively expressed in the preadipocyte cell line 3T3L1 and in primary preadipocytes isolated from murine mesenteric fat, while nucleotide oligomerization domain 2 was only weakly expressed by these cells. Treatment with lactyl-tetra-diaminopimelic acid, muramyl dipeptide, LPS, IL-1β, and TNF-α did not affect cellular nucleotide oligomerization domain 1 mRNA amounts. Except muramyl dipeptide, all factors significantly increased nucleotide oligomerization domain 2 mRNA in mesenteric fat preadipocytes after 4 h. However, specific stimulation of nucleotide oligomerization domain 1 induced IL-6 synthesis in preadipocytes from wild-type or TLR2/4-deficient mice. Confirming nucleotide oligomerization domain 1 specificity, transfection of nucleotide oligomerization domain 1-specific small interfering RNA significantly blocked the effect of lactyl-tetra-diaminopimelic acid on IL-6 production. With specific inhibitors and a NF-κB reporter plasmid, nucleotide oligomerization domain 1-mediated activation of NF-κB was shown to be responsible for the induction of IL-6 in preadipocytes. In addition, expression of functional nucleotide oligomerization domain 1 could be confirmed in primary human preadipocytes. In summary, we here identified preadipocytes as a novel cell population expressing nucleotide oligomerization domains 1 and 2. Not regulated on transcriptional level, nucleotide oligomerization domain 1 in preadipocytes serves as a sensor for bacterial degradation products and triggers proinflammatory effector responses. Thus, our results further strengthen the allocation of the mesenteric fat and especially of preadipocytes to the innate immune system.
CD43 Plays Both Antiadhesive and Proadhesive Roles in Neutrophil Rolling in a Context-Dependent Manner
Authors: Matsumoto Masanori , Shigeta Akiko , Miyasaka Masayuki , Hirata Takako
Published: April 2014

As the first step in the recruitment of neutrophils into tissues, the cells become tethered to and roll on the vessel wall. These processes are mediated by interactions between the P- and E-selectins, expressed on the endothelial cells of the vessel wall, and their ligands, expressed on the neutrophils. Recently, we reported that CD43 on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with P-selectin glycoprotein ligand-1 (PSGL-1), a major P- and E-selectin ligand. Here, we examined whether CD43 on neutrophils also functions as an E-selectin ligand. CD43 was precipitated with an E-selectin-IgG chimera from mouse bone marrow neutrophils. A CD43 deficiency diminished the E-selectin-binding activity of neutrophils when PSGL-1 was also deficient. Intravital microscopy showed that the CD43 deficiency significantly increased leukocyte rolling velocities in TNF-α-stimulated venules blocked with an anti-P-selectin mAb, where the rolling was mostly E-selectin dependent, when PSGL-1 was also absent. In contrast, in venules with trauma-induced inflammation, where the rolling was largely P-selectin dependent, the CD43 deficiency reduced leukocyte rolling velocities. Collectively, these observations suggest that CD43 generally serves as an antiadhesive molecule to attenuate neutrophil-endothelial interactions, but when E-selectin is expressed on endothelial cells, it also plays a proadhesive role as an E-selectin ligand.
Potent Simian Immunodeficiency Virus-Specific Cellular Immune Responses in the Breast Milk of Simian Immunodeficiency Virus-Infected, Lactating Rhesus Monkeys
Authors: Permar Sallie R. , Kang Helen H. , Carville Angela , Mansfield Keith G. , Gelman Rebecca S. , Rao Srinivas S. , Whitney James B. , Letvin Norman L.
Published: April 2014

Breast milk transmission of HIV is a leading cause of infant HIV/AIDS in the developing world. Remarkably, only a small minority of breastfeeding infants born to HIV-infected mothers contract HIV via breast milk exposure, raising the possibility that immune factors in the breast milk confer protection to the infants who remain uninfected. To model HIV-specific immunity in breast milk, lactation was pharmacologically induced in Mamu-A*01+ female rhesus monkeys. The composition of lymphocyte subsets in hormone-induced lactation breast milk was found to be similar to that in natural lactation breast milk. Hormone-induced lactating monkeys were inoculated i.v. with SIVmac251 and CD8+ T lymphocytes specific for two immunodominant SIV epitopes, Gag p11C and Tat TL8, and SIV viral load were monitored in peripheral blood and breast milk during acute infection. The breast milk viral load was 1–2 logs lower than plasma viral load through peak and set point of viremia. Surprisingly, whereas the kinetics of the SIV-specific cellular immunity in breast milk mirrored that of the blood, the peak magnitude of the SIV-specific CD8+ T lymphocyte response in breast milk was more than twice as high as the cellular immune response in the blood. Furthermore, the appearance of the SIV-specific CD8+ T lymphocyte response in breast milk was associated with a reduction in breast milk viral load, and this response remained higher than that in the blood after viral set point. This robust viral-specific cellular immune response in breast milk may contribute to control of breast milk virus replication.
Chronic Lymphocytic Leukemia Cells Bind and Present the Erythrocyte Protein Band 3: Possible Role as Initiators of Autoimmune Hemolytic Anemia
Authors: Galletti Jeremías , Cañones Cristian , Morande Pablo , Borge Mercedes , Oppezzo Pablo , Geffner Jorge , Bezares Raimundo , Gamberale Romina , Giordano Mirta
Published: April 2014

The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4+ T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.
Expression of a Soluble TGF-β Receptor by Tumor Cells Enhances Dendritic Cell/Tumor Fusion Vaccine Efficacy
Authors: Zhang Min , Berndt Bradford E. , Chen Jian-Jun , Kao John Y.
Published: April 2014

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-β limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-β on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-β receptor type II fused to the Fc region of human IgM (Adv-TGF-β-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-β receptor (TGF-β-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-β-R (4T1+Adv-TGF-β-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-β-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-β-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-β is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-β-R fusion vaccine induced fewer CD4+CD25+Foxp3+ Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4+CD25+ Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-β. We conclude that the blockade of tumor-derived TGF-β reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.
Inhalation of Staphylococcus aureus Enterotoxin A Induces IFN-γ and CD8 T Cell-Dependent Airway and Interstitial Lung Pathology in Mice
Authors: Muralimohan Guruprasaadh , Rossi Robert J. , Guernsey Linda A. , Thrall Roger S. , Vella Anthony T.
Published: April 2014

Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8+Vβ3+ T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-γ synthesis. The primary source of IFN-γ synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-γ deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-γ appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-γ-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.
The Src Kinase Lck Facilitates Assembly of HIV-1 at the Plasma Membrane
Authors: Strasner Amy B. , Natarajan Malini , Doman Tom , Key Douglas , August Avery , Henderson Andrew J.
Published: April 2014

HIV type 1 (HIV-1) assembly and egress are driven by the viral protein Gag and occur at the plasma membrane in T cells. Recent evidence indicates that secretory vesicles and machinery are essential components of virus packaging in both T cells and macrophages. However, the pathways and cellular mediators of Gag targeting to the plasma membrane are not well characterized. Lck, a lymphoid specific Src kinase critical for T cell activation, is found in the plasma membrane as well as various intracellular compartments and it has been suggested to influence HIV-1 replication. To investigate Lck as a potential regulator of Gag targeting, we assessed HIV-1 replication and Gag-induced virus-like particle release in the presence and absence of Lck. Release of HIV-1 and virus-like particles was reduced in the absence of Lck. This decrease in replication was not due to altered HIV-1 infection, transcription or protein translation. However, in T cells lacking Lck, HIV-1 accumulated intracellularly. In addition, expressing Lck in HeLa cells promoted HIV-1 Gag plasma membrane localization. Palmitoylation of the Lck unique domain, which is essential for directing Lck to the plasma membrane, was critical for its effect on HIV-1 replication. Furthermore, HIV-1 Gag directly interacted with the Lck unique domain in the context of infected cells. These results indicate that Lck plays a key role in targeting HIV-1 Gag to the plasma membrane in T cells.
Macrophage Polarization in Bacterial Infections
Authors: Benoit Marie , Desnues Benoît , Mege Jean-Louis
Published: April 2014

Converging studies have shown that M1 and M2 macrophages are functionally polarized in response to microorganisms and host mediators. Gene expression profiling of macrophages reveals that various Gram-negative and Gram-positive bacteria induce the transcriptional activity of a “common host response,” which includes genes belonging to the M1 program. However, excessive or prolonged M1 polarization can lead to tissue injury and contribute to pathogenesis. The so-called M2 macrophages play a critical role in the resolution of inflammation by producing anti-inflammatory mediators. These M2 cells cover a continuum of cells with different phenotypic and functional properties. In addition, some bacterial pathogens induce specific M2 programs in macrophages. In this review, we discuss the relevance of macrophage polarization in three domains of infectious diseases: resistance to infection, infectious pathogenesis, and chronic evolution of infectious diseases.
Cutting Edge: Programmed Death-1 Up-Regulation Is Involved in the Attrition of Cytomegalovirus-Specific CD8+ T Cells in Acute Self-Limited Hepatitis B Virus Infection
Authors: Zhang Ji-Yuan , Zhang Zheng , Jin Bo , Zhang Shu-Ye , Zhou Chun-Bao , Fu Jun-Liang , Wang Fu-Sheng
Published: April 2014

Attrition of heterologous virus-specific CD8+ T cells has been demonstrated in murine viral infection; however, little is known regarding this phenomenon in human viral infections. In this study, we observed that CMV-specific CD8+ T cells displayed numerical decline and functional impairment in the early phase of acute infection, whereas programmed death-1 (PD-1) expression was significantly up-regulated by these CMV-specific CD8+ T cells. This early PD-1 up-regulation was found to be closely associated with the increased apoptotic sensitivity of CMV-specific CD8+ T cells. The in vitro addition of anti-PD-1 further enhanced the spontaneous apoptosis of CMV-specific CD8+ T cells; however, blockade of the PD-1-mediated pathway with anti-PD-L1 significantly restored the CMV-specific CD8+ T cell proliferation and IFN-γ production. Thus, PD-1 plays a crucial role in the attrition of CMV-specific CD8+ T cells in acute hepatitis B virus infection, which in turn, influences the preexisting homeostatic virus-specific CD8+ T cell pool.
Cutting Edge: Instructive Role of Peripheral Tissue Cells in the Imprinting of T Cell Homing Receptor Patterns
Authors: Edele Fanny , Molenaar Rosalie , Gütle Dominique , Dudda Jan C. , Jakob Thilo , Homey Bernhard , Mebius Reina , Hornef Mathias , Martin Stefan F.
Published: April 2014

Tissue-specific homing of effector and memory T cells to skin and small intestine requires the imprinting of specific combinations of adhesion molecules and chemokine receptors by dendritic cells in the draining lymph nodes. In this study, we demonstrate that CD8+ T cells activated by Ag-pulsed bone marrow-derived dendritic cells were induced to express the small intestine homing receptors α4β7 integrin and chemokine receptor CCR9 in coculture with small intestinal epithelial cells. In contrast, in coculture with dermal fibroblasts the skin-homing receptor E-selectin ligand was induced. Interestingly, the imprinting of gut homing receptors on anti-CD3/anti-CD28 stimulated T cells was induced by soluble factors produced by small intestinal epithelial cells. Retinoic acid was identified as a crucial factor. These findings show that peripheral tissue cells directly produce homing receptor imprinting factors and suggest that dendritic cells can acquire their imprinting potential already in the peripheral tissue of origin.
Resistin Inhibits Essential Functions of Polymorphonuclear Leukocytes
Authors: Cohen Gerald , Ilic Dalibor , Raupachova Jana , Hörl Walter H.
Published: April 2014

The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion, resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.
Characterization of a Novel Nonclassical T Cell Clone with Broad Reactivity against Human Renal Cell Carcinomas
Authors: Wang Qiong J. , Hanada Ken-ichi , Yang James C.
Published: April 2014

A CD4+ T cell clone (HC/2G-1) was established by stimulating peripheral blood T cells from a patient with renal cell carcinoma (RCC) with dendritic cells preincubated with the autologous apoptotic renal tumor line in the presence of IFN-α. It recognizes the autologous RCC and most allogeneic RCC lines by IFN-γ release (10 of 11 lines) and lysis (9 of 10 lines), but does not recognize multiple EBV B cells or fibroblasts. It shows little or no recognition of a panel of melanomas, breast cancers and non-small–cell lung cancers. Phenotypically, HC/2G-1 is CD3+CD4+ TCR αβ+, but CD161−CD16−NKG2D−. Tumor recognition by clone HC/2G-1 was not blocked by Abs to HLA class I or class II, but was significantly reduced by anti-TCR αβ Ab. Furthermore, tumor recognition was β2-microglobulin-independent. HC/2G-1 does not use a Vα or Vβ described for classical NKT cells, but rather Vα14 and Vβ2.1. Allogeneic T cells cotransfected with mRNAs encoding the α and β chains of the HC/2G-1 TCR recognized renal tumor lines, demonstrating that tumor recognition is TCR-mediated. Interestingly, TRAIL appears to play a role in tumor recognition by HC/2G-1 in that reactivity was blocked by anti-TRAIL Ab, and soluble TRAIL could enhance IFN-γ secretion by HC/2G-1 in response to renal tumors. Our findings suggest that clone HC/2G-1 represents a novel type of CD4+ cell that has broad TCR-mediated recognition of a determinant widely expressed by RCC.
Rab27a Regulates Exocytosis of Tertiary and Specific Granules in Human Neutrophils
Authors: Herrero-Turrión M. Javier , Calafat Jero , Janssen Hans , Fukuda Mitsunori , Mollinedo Faustino
Published: April 2014

The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinase-enriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca2+ and GTP-γ-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation.
MyD88 Plays a Critical T Cell-Intrinsic Role in Supporting CD8 T Cell Expansion during Acute Lymphocytic Choriomeningitis Virus Infection
Authors: Rahman Adeeb H. , Cui Weiguo , LaRosa David F. , Taylor Devon K. , Zhang Jidong , Goldstein Daniel R. , Wherry E. John , Kaech Susan M. , Turka Laurence A.
Published: April 2014

During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88−/− CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.
Cell Cycle-Related Acquisition of Cytotoxic Mediators Defines the Progressive Differentiation to Effector Status for Virus-Specific CD8+ T Cells
Authors: Jenkins Misty R. , Mintern Justine , La Gruta Nicole L. , Kedzierska Katherine , Doherty Peter C. , Turner Stephen J.
Published: April 2014

Although analysis of virus-specific CTL function at the peak of infection suggests that granzyme (grz) and perforin (pfp) gene expression is not coregulated, early differentiation events leading to acquisition of function are poorly understood. Using a combination of CFSE dilutions and single-cell RT-PCR, effector gene expression was determined early after CTL activation. There were low levels of pfp and grz expression at division 3, with increased expression by divisions 6–8. The increase in effector mRNA expression with division correlated with increasing ex vivo cytotoxicity. Of the mRNA transcripts detected at division 3, there was an increased frequency of grzB and grzK (compared with grzA or pfp), and this pattern was also observed at later divisions. The prevalence of OT-I CTL expressing grz/pfp mRNA was equivalent for the divided CD62Lhigh and CD62Llow sets, but the concentrations of grzB protein, levels of CTL activity, and the absolute amounts of grzB transcript were substantially greater for the CD62Llow population. Thus, while effector gene expression can be acquired early, maturation of cytotoxic capacity requires extended differentiation.
B Cell Receptor Affinity and B Cell Subset Identity Integrate to Define the Effectiveness, Affinity Threshold, and Mechanism of Anergy
Authors: Diz Ramiro , McCray Suzanne K. , Clarke Stephen H.
Published: April 2014

In this study we show that BCR affinity and subset identity make unique contributions to anergy. Analysis of anti-Smith (Sm) B cells of different affinities indicates that increasing affinity improves anergy’s effectiveness while paradoxically increasing the likelihood of marginal zone (MZ) and B-1 B cell differentiation rather than just follicular (FO) B cell differentiation. Subset identity in turn determines the affinity threshold and mechanism of anergy. Subset-specific affinity thresholds for anergy induction allow discordant regulation of low-affinity anti-Sm FO and MZ B cells and could account for the higher frequency of autoreactive MZ B cells than that of FO B cells in normal mice. The mechanism of anergy changes during differentiation and differs between subsets. This is strikingly illustrated by the observation that blockade of BCR-mediated activation of FO and MZ B cells occurs at different levels in the signaling cascade. Thus, attributes unique to B cells of each subset integrate with signals from the BCR to determine the effectiveness, affinity threshold, and mechanism of anergy.
IgE-Induced Mast Cell Survival Requires the Prolonged Generation of Reactive Oxygen Species
Authors: Sly Laura M. , Kalesnikoff Janet , Lam Vivian , Wong Dana , Song Christine , Omeis Stephanie , Chan Karen , Lee Corinna W. K. , Siraganian Reuben P. , Rivera Juan , Krystal Gerald
Published: April 2014

We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca2+) entry. However, whereas IgE+Ag more potently stimulates Ca2+ entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca2+ entry than IgE+Ag initially, they all trigger a more prolonged Ca2+ influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca2+ influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H2O2 to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.
Cytokine-Mediated Inhibition of Fibrillar Amyloid-β Peptide Degradation by Human Mononuclear Phagocytes
Authors: Yamamoto Masaru , Kiyota Tomomi , Walsh Shannon M. , Liu Jianuo , Kipnis Jonathan , Ikezu Tsuneya
Published: April 2014

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-β peptides (Aβ) in brain. Although Aβ uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Aβ degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Aβ using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer 125I-Aβ40 and Aβ42. Initial uptake of fibrillar Aβ40 and Aβ42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Aβ less than 0.5% and saturation of degradation within 24 h. IFN-γ increased the intracellular retention of fibrillar Aβ40 and Aβ42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-β1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Aβ degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-γ and TNF-α directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Aβ degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Aβ degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.
Fibroblast-Type Reticular Stromal Cells Regulate the Lymph Node Vasculature
Authors: Chyou Susan , Ekland Eric H. , Carpenter April C. , Tzeng Te-Chen Jenny , Tian Sha , Michaud Michael , Madri Joseph A. , Lu Theresa T.
Published: April 2014

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)+ endothelial cells are less sensitive than PNAd− endothelial cells to VEGF blockade. Lymphotoxin β receptor (LTβR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTβR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTβR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd+ and PNAd− endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.