bluuebunny/crossref_metadata_2025 huggingface dataset:
Aluminum Hydroxide Adjuvants Activate Caspase-1 and Induce IL-1β and IL-18 Release
Authors: Li Hanfen , Nookala Suba , Re Fabio
Published: April 2014

Aluminum hydroxide (Alum) is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. In this study, we show that Alum activates caspase-1 and induce secretion of mature IL-1β and IL-18. Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists released only traces of IL-1β or IL-18, despite the fact that the IL-1β mRNA was readily induced by both TLR agonists. In contrast, cells costimulated with TLR agonists plus Alum released large amount of IL-1β and IL-18. Alum-induced IL-1β and IL-18 production was not due to enhancement of TLR signaling but rather reflected caspase-1 activation and in mouse dendritic cells occurred in a MyD88-independent fashion. Secretion of other proinflammatory cytokines such as IL-8 was not affected by Alum treatments. However, TLR-induced production of IL-10 was increased and that of IFN-γ-inducible protein decreased by Alum cotreatment. Considering the immunostimulatory activities of these cytokines and the ability of IL-1β to act as adjuvant, our results suggest a mechanism for the adjuvanticity of Alum.
Inflammatory Reactive Oxygen Species-Mediated Hemopoietic Suppression in Fancc-Deficient Mice
Authors: Sejas Daniel P. , Rani Reena , Qiu Yuhui , Zhang Xiaoling , Fagerlie Sara R. , Nakano Hiroyasu , Williams David A. , Pang Qishen
Published: April 2014

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow (BM) failure and have a high risk of cancer. Certain manifestations of the disease suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both BM failure and malignancies. In this study, we have investigated inflammation and innate immunity in FA hemopoietic cells using mice deficient in Fanconi complementation group C gene (Fancc). We demonstrate that Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to LPS-induced septic shock as a result of hemopoietic suppression. This exacerbated inflammatory phenotype is intrinsic to the hemopoietic system and can be corrected by the re-expression of a wild-type FANCC gene, suggesting a potential role of the FANCC protein in innate immunity. LPS-mediated hemopoietic suppression requires two major inflammatory agents, TNF-α and reactive oxygen species. In addition, LPS-induced excessive accumulation of reactive oxygen species in Fancc−/− BM cells overactivates the stress kinase p38 and requires prolonged activation of the JNK. Our data implicate a role of inflammation in pathogenesis of FA and BM failure diseases in general.
Scar-Associated Macrophages Are a Major Source of Hepatic Matrix Metalloproteinase-13 and Facilitate the Resolution of Murine Hepatic Fibrosis
Authors: Fallowfield Jonathan A. , Mizuno Masashi , Kendall Timothy J. , Constandinou Christothea M. , Benyon R. Christopher , Duffield Jeremy S. , Iredale John P.
Published: April 2014

Both the identity and source of the rodent collagenase(s) that mediates matrix remodeling in liver fibrosis remain elusive. We have recently demonstrated an unequivocal role for scar-associated macrophages (SAMs) in the spontaneous resolution of liver fibrosis and sought to determine whether SAMs are the source of matrix metalloproteinase (MMP) 13 (collagenase 3), considered to be the primary interstitial collagenase in rodents. In this study, we demonstrate an association between MMP13 expression and the presence of SAMs in the regression of experimental liver fibrosis. mmp13 gene expression was restricted to regions of fibrosis that were rich in SAMs. Both MMP13 mRNA and protein colocalized to large phagocytes within and directly apposed to hepatic scars. Using the CD11b-DTR-transgenic mouse to deplete SAMs in a model of chronic CCl4 injury, we found that SAM depletion resulted in a 5-fold reduction in mmp13 message (p = 0.005). Furthermore, resolution of CCl4-induced fibrosis was retarded in MMP13-deficient mice. Thus, SAMs selectively, during resolution of fibrosis induce and use the major collagenase MMP13 to mediate the resorption of interstitial matrix and successfully remodel the fibrotic liver.
Some Plasmin-Induced Antibodies Bind to Cardiolipin, Display Lupus Anticoagulant Activity and Induce Fetal Loss in Mice
Authors: Chen Xiao-Xiang , Gu Yue-Ying , Li Shu-Jie , Qian Jie , Hwang Kwan-Ki , Chen Pojen P. , Chen Shun-Le , Yang Cheng-De
Published: April 2014

The combined presence of anti-phospholipid Ab (aPL), thrombosis, and/or fetal loss is recognized as the antiphospholipid syndrome (APS). aPL include anti-cardiolipin Ab (aCL) and/or lupus anticoagulants (LAC, detected as Ig that prolong certain in vitro phospholipid (PL)-restricted blood clotting tests); both aCL and LAC are the diagnostic Ab for APS. Studies show that aPL represent a heterogeneous group of Ab, which recognize various PL, PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found that five of seven patient-derived IgG monoclonal aCL react with thrombin, activated protein C, and plasmin. All three proteins are trypsin-like serine proteases (SP), and are highly homologous in their catalytic domains. Importantly, among these SP autoantigens, the reactive aCL bind to plasmin with the highest affinity, suggesting that plasmin may serve as a major driving autoantigen for some aCL in ∼30% of APS patients who are positive for IgG anti-plasmin Ab. To test this hypothesis, we immunized BALB/c mice with human plasmin and analyzed immune sera for aCL activity and reactivity with relevant SP. We found that some immune sera displayed aCL activity and/or bound to test SP. Subsequently, eight mAb were obtained and studied. The results revealed that one mAb displayed the aCL and the LAC activities and induced fetal loss when injected into pregnant mice. Immunohistological analyses of placentas revealed extensive deposits of activated C3 components. Combined, these data demonstrate that plasmin may serve as a driving Ag for some pathogenic aPL.
Unequal Contribution of Akt Isoforms in the Double-Negative to Double-Positive Thymocyte Transition
Authors: Mao Changchuin , Tili Esmerina G. , Dose Marei , Haks Mariëlle C. , Bear Susan E. , Maroulakou Ioanna , Horie Kyoji , Gaitanaris George A. , Fidanza Vincenzo , Ludwig Thomas , Wiest David L. , Gounari Fotini , Tsichlis Philip N.
Published: April 2014

Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.
CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation
Authors: Prechtel Alexander T. , Turza Nadine M. , Theodoridis Alexandros A. , Steinkasserer Alexander
Published: April 2014

Mature human dendritic cells (mDCs) are the most powerful APCs known today, having the unique ability to induce primary immune responses. One of the best known surface markers for mDCs is the glycoprotein CD83, which is strongly up-regulated during maturation, together with costimulatory molecules such as CD80 and CD86. When CD83 surface expression was inhibited by interference with the messenger RNA export or by infection with certain viruses, DCs showed a dramatically reduced capability to induce T cell proliferation. However, in these cases side effects on other cellular functions cannot be excluded completely. In this study we present an efficient method to specifically influence CD83 surface expression by the use of RNA interference. We used small-interfering RNA targeted against CD83 and carefully evaluated an electroporation protocol for the delivery of the duplex into the cells. Furthermore, we identified freshly prepared immature DCs as the best target for the application of a CD83 knockdown and we were also able to achieve a long lasting silencing effect for this molecule. Finally, we were able to confirm that CD83 functions as an enhancer during the stimulation of T cells, significantly increases DC-mediated T cell proliferation, and goes hand in hand with clear changes in cytokine expression during T cell priming. These results were obtained for the first time without the use of agents that might cause unwanted side effects, such as low m.w. inhibitors or viruses. Therefore, this method presents a suitable way to influence DC biology.
Down-Regulation of IL-7Rα Expression in Human T Cells via DNA Methylation
Authors: Kim Hang-Rae , Hwang Kyung-A , Kim Ki-Chan , Kang Insoo
Published: April 2014

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Rαhigh and IL-7Rαlow with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Rα gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Rα gene expression in human CD8+ T cells and Jurkat T cells. IL-7RαhighCD8+ T cells had decreased methylation in the IL-7Rα gene promoter compared with IL-7RαlowCD8+ T cells and Jurkat T cells with low levels of IL-7Rα. Treating Jurkat T cells with 5-aza-2′-deoxycytidine, which reduced DNA methylation, increased IL-7Rα expression. Plus, the unmethylated IL-7Rα gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Rα expression in T cells via affecting IL-7Rα gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.
Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling
Authors: Sanderson Joseph P. , Naisbitt Dean J. , Farrell John , Ashby Charlotte A. , Tucker M. Jane , Rieder Michael J. , Pirmohamed Munir , Clarke Stephen E. , Park B. Kevin
Published: April 2014

Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250–500 μM) and the protein-reactive metabolite nitroso sulfamethoxazole (1–10 μM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.
CTLA-4 Differentially Regulates the Immunological Synapse in CD4 T Cell Subsets
Authors: Jackman Rachael P. , Balamuth Fran , Bottomly Kim
Published: April 2014

Primary murine Th1 and Th2 cells differ in the organization of the immunological synapse, with Th1 cells, but not Th2 cells, clustering signaling molecules at the T cell/B cell synapse site. We sought to determine whether differential costimulatory signals could account for the differences observed. We found that Th2 cells express higher levels of CTLA-4 than Th1 cells, and demonstrated that Th2 cells lacking CTLA-4 are now able to cluster the TCR with the same frequency as Th1 cells. Furthermore, reconstitution of CTLA-4 into CTLA-4-deficient Th2 cells, or into Th1 cells, inhibits the clustering of the TCR. We have also shown that Th2 cells, but not Th1 cells, show variations in the organization of the immunological synapse depending on levels of expression of CD80/CD86 on the APC. These studies demonstrate a unique role for CTLA-4 as a critical regulator of Th2 cells and the immunological synapse.
Inhibitory Receptor Signals Suppress Ligation-Induced Recruitment of NKG2D to GM1-Rich Membrane Domains at the Human NK Cell Immune Synapse
Authors: Endt Johanna , McCann Fiona E. , Almeida Catarina R. , Urlaub Doris , Leung Rufina , Pende Daniela , Davis Daniel M. , Watzl Carsten
Published: April 2014

NKG2D is an activating receptor expressed on all human NK cells and a subset of T cells. In cytolytic conjugates between NK cells and target cells expressing its ligand MHC class I chain-related gene A, NKG2D accumulates at the immunological synapse with GM1-rich microdomains. Furthermore, NKG2D is specifically recruited to detergent-resistant membrane fractions upon ligation. However, in the presence of a strong inhibitory stimulus, NKG2D-mediated cytotoxicity can be intercepted, and recruitment of NKG2D to the immunological synapse and detergent-resistant membrane fractions is blocked. Also, downstream phosphorylation of Vav-1 triggered by NKG2D ligation is circumvented by coengaging inhibitory receptors. Thus, we propose that one way in which inhibitory signaling can control NKG2D-mediated activation is by blocking its recruitment to GM1-rich membrane domains. The accumulation of activating NK cell receptors in GM1-rich microdomains may provide the necessary platform from which stimulatory signals can proceed.
Quantitatively Reduced Participation of Anti-Nuclear Antigen B Cells That Down-Regulate B Cell Receptor during Primary Development in the Germinal Center/Memory B Cell Response to Foreign Antigen
Authors: Alabyev Boris , Rahman Ziaur S. M. , Manser Tim
Published: April 2014

The peripheral B cell compartment contains high levels of “polyreactivity” including autospecificities. We have described a pathway that certain autoreactive B cells may take in gaining stable access to the foreign Ag-responsive peripheral compartment. This pathway was revealed in mice expressing a targeted Ig H chain transgene encoding BCRs with “multireactivity” for the hapten arsonate and DNA-based autoantigens. B cells expressing such BCRs develop to mature follicular phenotype and locale, and are not short-lived. These B cells express very low levels of BCR, indicating that they are not “ignorant” of self Ag, but do not display features of anergy in in vitro assays. Nonetheless, a variety of states of lymphocyte anergy has been described, and some may only be manifested in vivo. As such, we analyzed the ability of these B cells to participate in a T cell-dependent immune response to arsonate in vivo. These B cells mount an early primary response similar to control B cells, including homing to follicles, migration to the T-B interface, and induction of costimulatory molecules, proliferation, differentiation to AFCs, class switching, and entry into GCs and somatic hypermutation. Nonetheless, these B cells display reduced participation in the latter stages of the GC response and in the anamnestic AFC response. In total, these data suggest that while the autoreactivity of this type of B cell does not result in anergy, the ability of such B cells to participate in a cross-reactive immune response to foreign Ag is compromised.
Tertiary Lymphoid Structures in the Pancreas Promote Selection of B Lymphocytes in Autoimmune Diabetes
Authors: Kendall Peggy L. , Yu Guowu , Woodward Emily J. , Thomas James W.
Published: April 2014

Autoimmune diabetes occurs when invading lymphocytes destroy insulin-producing β cells in pancreatic islets. The role of lymphocytic aggregates at this inflammatory site is not understood. We find that B and T lymphocytes attacking islets in NOD mice organize into lymphoid structures with germinal centers. Analysis of BCR L chain genes was used to investigate selection of B lymphocytes in these tertiary lymphoid structures and in draining pancreatic lymph nodes. The pancreatic repertoire as a whole was found to be highly diverse, with the profile of L chain genes isolated from whole pancreas differing from that observed in regional lymph nodes. A Vκ14 L chain predominated within the complex pancreatic repertoire of NOD mice. Skewing toward Vκ4 genes was observed in the pancreas when the repertoire of NOD mice was restricted using a fixed Ig H chain transgene. Nucleotide sequencing of expressed Vκs identified shared mutations in some sequences consistent with Ag-driven selection and clonal expansion at the site of inflammation. Isolated islets contained oligoclonal B lymphocytes enriched for the germinal center marker GL7 and for sequences containing multiple mutations within CDRs, suggesting local T-B interactions. Together, these findings identify a process that selects B lymphocyte specificities within the pancreas, with further evolution of the selected repertoire at the inflamed site. This interpretation is reinforced by Ag-binding studies showing a large population of insulin-binding B lymphocytes in the pancreas compared with draining lymph nodes.
Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells
Authors: Mummidi Srinivas , Adams Lisa M. , VanCompernolle Scott E. , Kalkonde Mrunal , Camargo Jose F. , Kulkarni Hemant , Bellinger Adam S. , Bonello Gregory , Tagoh Hiromi , Ahuja Seema S. , Unutmaz Derya , Ahuja Sunil K.
Published: April 2014

Surface levels of CCR5 on memory CD4+ T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.
Regulation of αβ/γδ T Cell Development by the Activator Protein 1 Transcription Factor c-Jun
Authors: Riera-Sans Lluís , Behrens Axel
Published: April 2014

c-Jun is a member of the AP-1 family of transcription factors, the activity of which is strongly augmented by TCR signaling. To elucidate the functions of c-Jun in mouse thymic lymphopoiesis, we conditionally inactivated c-Jun specifically during early T cell development. The loss of c-Jun resulted in enhanced generation of γδ T cells, whereas αβ T cell development was partially arrested at the double-negative 3 stage. The increased generation of γδ T cells by loss of c-Jun was cell autonomous, because in a competitive reconstitution experiment the knockout-derived cells produced more γδ T cells than did the control cells. C-jun-deficient immature T cells failed to efficiently repress transcription of IL-7Rα, resulting in augmented IL-7Rα mRNA and surface levels. Chromatin immunoprecipitation assays revealed binding of c-Jun to AP-1 binding sites present in the IL-7Rα promoter, indicating direct transcriptional regulation. Thus, c-Jun controls the transcription of IL-7Rα and is a novel regulator of the αβ/γδ T cell development.
Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection
Authors: Rydström Anna , Wick Mary Jo
Published: April 2014

Neutrophils, monocytes, and dendritic cells (DC) are phenotypically and functionally related phagocytes whose presence in infected tissues is critical to host survival. Their overlapping expression pattern of surface molecules, the differentiation capacity of monocytes, and the presence of monocyte subsets underscores the complexity of understanding the role of these cells during infection. In this study we use five- to seven-color flow cytometry to assess the phenotype and function of monocytes recruited to Peyer’s patches (PP) and mesenteric lymph nodes (MLN) after oral Salmonella infection of mice. The data show that CD68highGr-1int (intermediate) monocytes, along with CD68intGr-1high neutrophils, rapidly accumulate in PP and MLN. The monocytes have increased MHC-II and costimulatory molecule expression and, in contrast to neutrophils and DC, produce inducible NO synthase. Although neutrophils and monocytes from infected mice produce TNF-α and IL-1β upon ex vivo culture, DC do not. In addition, although recruited monocytes internalize Salmonella in vitro and in vivo they did not induce the proliferation of OT-II CD4+ T cells after coincubation with Salmonella expressing OVA despite their ability to activate OT-II cells when pulsed with the OVA323–339 peptide. We also show that recruited monocytes enter the PP of infected mice independently of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Finally, recruited but not resident monocytes increase in the blood of orally infected mice, and MHC-II up-regulation, but not TNF-α or iNOS production, occur already in the blood. These studies are the first to describe the accumulation and function of monocyte subsets in the blood and GALT during oral Salmonella infection.
MyD88-Mediated Instructive Signals in Dendritic Cells Regulate Pulmonary Immune Responses during Respiratory Virus Infection
Authors: Rudd Brian D. , Schaller Matthew A. , Smit Joost J. , Kunkel Steven L. , Neupane Rupak , Kelley Lara , Berlin Aaron A. , Lukacs Nicholas W.
Published: April 2014

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88−/− mice. The exacerbation of RSV pathophysiology in MyD88−/− mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88−/− mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88−/− BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-γ from CD4+ T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88−/− mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88−/− BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.
Lung-Specific Overexpression of CC Chemokine Ligand (CCL) 2 Enhances the Host Defense to Streptococcus pneumoniae Infection in Mice: Role of the CCL2-CCR2 Axis
Authors: Winter Christine , Taut Katharina , Srivastava Mrigank , Länger Florian , Mack Matthias , Briles David E. , Paton James C. , Maus Regina , Welte Tobias , Gunn Michael D. , Maus Ulrich A.
Published: April 2014

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.
Induction of IL-13 Triggers TGF-β1-Dependent Tissue Fibrosis in Chronic 2,4,6-Trinitrobenzene Sulfonic Acid Colitis
Authors: Fichtner-Feigl Stefan , Fuss Ivan J. , Young Cheryl A. , Watanabe Tomohiro , Geissler Edward K. , Schlitt Hans-Jürgen , Kitani Atsushi , Strober Warren
Published: April 2014

To investigate the immunopathogenesis of inflammation-associated fibrosis, we analyzed the chronic colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intrarectal 2,4,6-trinitrobenzene sulfonic acid. We showed first in this model that an initial Th1 response involving IL-12p70 and IFN-γ subsides after 3 wk to be supplanted by an IL-23/IL-25 response beginning after 4–5 wk. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a Th2 response, particularly IL-13, which reaches a plateau at 8–9 wk. In vitro stimulation studies suggest that this IL-13 production is dependent on IL-23 and IL-25, but not on IL-12p70. We then show that IL-13 production results in the induction of an IL-13R formerly thought to function only as a decoy receptor, IL-13Rα2, and this receptor is critical to the production of TGF-β1 and the onset of fibrosis. Thus, if IL-13 signaling through this receptor is blocked by administration of soluble IL-13Rα2-Fc, or by administration of IL-13Rα2-specific small interfering RNA, TGF-β1 is not produced and fibrosis does not occur. These studies show that in chronic 2,4,6-trinitrobenzene sulfonic acid colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell surface-expressed IL-13R to induce TGF-β1. A similar mechanism may obtain in certain forms of human inflammatory bowel disease.
Scavenger Receptors SR-AI/II and MARCO Limit Pulmonary Dendritic Cell Migration and Allergic Airway Inflammation
Authors: Arredouani Mohamed S. , Franco Francesca , Imrich Amy , Fedulov Alexey , Lu Xin , Perkins David , Soininen Raija , Tryggvason Karl , Shapiro Steven D. , Kobzik Lester
Published: April 2014

The class A scavenger receptors (SR-A) MARCO and SR-AI/II are expressed on lung macrophages (MΦs) and dendritic cells (DCs) and function in innate defenses against inhaled pathogens and particles. Increased expression of SR-As in the lungs of mice in an OVA-asthma model suggested an additional role in modulating responses to an inhaled allergen. After OVA sensitization and aerosol challenge, SR-AI/II and MARCO-deficient mice exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness compared with wild-type mice. A role for simple SR-A-mediated Ag clearance (“scavenging”) by lung MΦs was excluded by the observation of a comparable uptake of fluorescent OVA by wild-type and SR-A-deficient lung MΦs and DCs. In contrast, airway instillation of fluorescent Ag revealed a significantly higher traffic of labeled DCs to thoracic lymph nodes in SR-A-deficient mice than in controls. The increased migration of SR-A-deficient DCs was accompanied by the enhanced proliferation in thoracic lymph nodes of adoptively transferred OVA-specific T cells after airway OVA challenge. The data identify a novel role for SR-As expressed on lung DCs in the down-regulation of specific immune responses to aeroallergens by the reduction of DC migration from the site of Ag uptake to the draining lymph nodes.
Genetic Stability of Human T Lymphotropic Virus Type I despite Antiviral Pressures by CTLs
Authors: Kubota Ryuji , Hanada Kousuke , Furukawa Yoshitaka , Arimura Kimiyoshi , Osame Mitsuhiro , Gojobori Takashi , Izumo Shuji
Published: April 2014

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease. Patients with HAM/TSP show high proviral load despite increased HTLV-I Tax-specific CTL. It is still unknown whether the CTL efficiently eliminate the virus in vivo and/or whether a naturally occurring variant virus becomes predominant by escaping from the CTL. To address these issues, we sequenced a large number of HTLV-I tax genes from HLA-A*02 HAM/TSP patients and estimated synonymous and nonsynonymous changes of the genes to detect positive selection pressure on the virus. We found the pressures in three of six CTL epitopes in HTLV-I Tax, where amino acid substitutions preferentially occurred. Although some of variant viruses were not recognized by the CTL, no variant viruses accumulated within 3–8 years, indicating genetic stability of HTLV-I tax gene. These results suggest that CTL eliminate the infected cells in vivo and naturally occurring variant viruses do not predominate. As Tax is a regulatory protein which controls viral replication, the amino acid substitutions in Tax may reduce viral fitness for replication. Viral fitness and host immune response may contribute to the viral evolution within the infected individuals. Furthermore, the genetic stability in the epitopes despite the antiviral pressures suggests that the three epitopes can be the candidate targets for HTLV-I vaccine development.
A Key Role for Itk in Both IFNγ and IL-4 Production by NKT Cells
Authors: Au-Yeung Byron B. , Fowell Deborah J.
Published: April 2014

NKT cells rapidly secrete cytokines upon TCR stimulation and thus may modulate the acquired immune response. Recent studies suggest that signaling for development and effector function in NKT cells may differ from conventional T cells. The tyrosine kinase Itk is activated downstream of the TCR, and its absence in CD4+ T cells results in impaired Th2, but not Th1 responses. In this study, we investigated NKT cell function in the absence of Itk as impaired type 2 responses in vivo could be manifest through IL-4 defects in a number of cell types. We show that Itk-deficient NKT cells up-regulate IL-4 mRNA in the thymus and express constitutive IL-4 and IFN-γ transcripts in peripheral organs. Thus, Itk is not required for the developmental activation of cytokine loci in NKT cells. Nevertheless, Itk-deficient NKT cells are severely impaired in IL-4 protein production. Strikingly, unlike conventional CD4+ T cells, Itk-deficient NKT cells also have profound defects in IFN-γ production. Furthermore, both IL-4 and IFN-γ production were markedly impaired following in vivo challenge with α-galactosyl ceramide. Function can be restored in Itk-deficient NKT cells by provision of calcium signals using ionomycin. These results suggest that NKT cells are highly dependent on Itk for IL-4- and IFN-γ-mediated effector function. Thus, the pattern of cytokine genes that are affected by Itk deficiency appears to be cell lineage-specific, likely reflecting differences in activation threshold between immune effectors. The severe defect in NKT cell function may underlie a number of the Th1 and Th2 immune defects in Itk-deficient mice.
Human IgM+CD27+ B Cells: Memory B Cells or “Memory” B Cells?
Authors: Tangye Stuart G. , Good Kim L.
Published: April 2014

Memory B cells are generated in germinal centers (GC) and contribute to serological immunity by rapidly differentiating into plasma cells. Human memory B cells can be identified by the expression of CD27. These cells exhibit more rapid responses than naive (CD27−) B cells following stimulation in vitro, consistent with the heightened kinetics of secondary responses in vivo. CD27+ B cells express mutated Ig V region genes; however a significant proportion continue to express IgM, suggesting the existence of IgM+ memory B cells. The observation that mutated IgM+CD27+ B cells are generated in humans who cannot form GC led to the conclusions that these cells are generated independently of GC and thus are not memory cells and that they mediate responses to T cell-independent Ag. Although some studies support the idea that IgM+CD27+ B cells participate in T cell-independent responses, many others do not. In this review we will provide alternate interpretations of the biology of IgM+CD27+ B cells and propose that they are indeed memory cells.
Interaction of TNF with TNF Receptor Type 2 Promotes Expansion and Function of Mouse CD4+CD25+ T Regulatory Cells
Authors: Chen Xin , Bäumel Monika , Männel Daniela N. , Howard O. M. Zack , Oppenheim Joost J.
Published: April 2014

Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4+CD25+ T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4+CD25− T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1β and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2−/− mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.
Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Modifies T Cells at Multiple Steps and Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis
Authors: Brahmachari Saurav , Pahan Kalipada
Published: April 2014

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis.
Proteomic Analysis of the Inflamed Intestinal Mucosa Reveals Distinctive Immune Response Profiles in Crohn’s Disease and Ulcerative Colitis
Authors: Berndt Uta , Bartsch Sebastian , Philipsen Lars , Danese Silvio , Wiedenmann Bertram , Dignass Axel U. , Hämmerle Marcus , Sturm Andreas
Published: April 2014

Although Crohn’s disease (CrD) and ulcerative colitis (UC) share several clinical features, the mechanisms of tissue injury differ. Because the global cellular function depends upon the protein network environment as a whole, we explored changes in the distribution and association of mucosal proteins to define key events involved in disease pathogenesis. Endoscopic biopsies were taken from CrD, UC, and control colonic mucosa, and Multi-Epitope-Ligand-Cartographie immunofluorescence microscopy with 32 different Abs was performed. Multi-Epitope-Ligand-Cartographie is a novel, highly multiplexed robotic imaging technology which allows integrating cell biology and biomathematical tools to visualize dozens of proteins simultaneously in a structurally intact cell or tissue. In CrD, the number of CD3+CD45RA+ naive T cells was markedly increased, but only activated memory, but not naive, T cells expressed decreased levels of Bax, active caspase-3 or -8. In UC, only CD4+ T cells coexpressing NF-κB were caspase-8 and poly(ADP-ribose)-polymerase positive. Furthermore, the number of CD4+CD25+ T cells was elevated only in UC, whereas in CrD and controls, the number of these cells was similar. By using hub analysis, we also identified that the colocalization pattern with NF-κB+ and poly(ADP-ribose)-polymerase+ as base motifs distinguished CrD from UC. High-content proteomic analysis of the intestinal mucosa demonstrated for the first time that different T cell populations within the intestinal mucosa express proteins translating distinct biological functions in each form of inflammatory bowel disease. Thus, topological proteomic analysis may help to unravel the pathogenesis of inflammatory bowel disease by defining distinct immunopathogenic profiles in CrD and UC.
Cutting Edge: Natural DNA Repetitive Extragenic Sequences from Gram-Negative Pathogens Strongly Stimulate TLR9
Authors: Magnusson Mattias , Tobes Raquel , Sancho Jaime , Pareja Eduardo
Published: April 2014

Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-α production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9tm1Aki knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.
Deficiency of Fatty Acid-Binding Proteins in Mice Confers Protection from Development of Experimental Autoimmune Encephalomyelitis
Authors: Reynolds Joseph M. , Liu Qiaohong , Brittingham Katherine C. , Liu Yawei , Gruenthal Michael , Gorgun Cem Z. , Hotamisligil Gökhan S. , Stout Robert D. , Suttles Jill
Published: April 2014

Fatty acid-binding proteins (FABPs) act as intracellular receptors for a variety of hydrophobic compounds, enabling their diffusion within the cytoplasmic compartment. Recent studies have demonstrated the ability of FABPs to simultaneously regulate metabolic and inflammatory pathways. We investigated the role of adipocyte FABP and epithelial FABP in the development of experimental autoimmune encephalomyelitis to test the hypothesis that these FABPs impact adaptive immune responses and contribute to the pathogenesis of autoimmune disease. FABP-deficient mice exhibited a lower incidence of disease, reduced clinical symptoms of experimental autoimmune encephalomyelitis and dramatically lower levels of proinflammatory cytokine mRNA expression in CNS tissue as compared with wild-type mice. In vitro Ag recall responses of myelin oligodendrocyte glycoprotein 35–55-immunized FABP−/− mice showed reduced proliferation and impaired IFN-γ production. Dendritic cells deficient for FABPs were found to be poor producers of proinflammatory cytokines and Ag presentation by FABP−/− dendritic cells did not promote proinflammatory T cell responses. This study reveals that metabolic-inflammatory pathway cross-regulation by FABPs contributes to adaptive immune responses and subsequent autoimmune inflammation.
Altered Dynamics of Kv1.3 Channel Compartmentalization in the Immunological Synapse in Systemic Lupus Erythematosus
Authors: Nicolaou Stella A. , Szigligeti Peter , Neumeier Lisa , Molleran Lee Susan , Duncan Heather J. , Kant Shashi K. , Mongey Anne Barbara , Filipovich Alexandra H. , Conforti Laura
Published: April 2014

Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca2+ influx. Although Kv1.3 channels have such an important role in T cell function, their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1–30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells, the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells, but the K channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.
Cutting Edge: Migration to Nonlymphoid Tissues Results in Functional Conversion of Central to Effector Memory CD8 T Cells
Authors: Marzo Amanda L. , Yagita Hideo , Lefrançois Leo
Published: April 2014

Memory CD8 T cells, essential for defense against intracellular pathogens, are heterogeneous with respect to phenotype and function. Constitutively lytic effector memory cells primarily reside in nonlymphoid tissues, whereas secondary lymphoid tissues contain functionally quiescent central memory cells. However, the mechanism by which functionally distinct memory populations are maintained is unknown. In this study, we show that resting CD8 memory cells modified their functional abilities upon entry into nonlymphoid tissues, as exemplified by the induction of granzyme B and lytic activity. Contemporaneously, the costimulator CD27 was down-regulated. These findings hold important implications for memory cell lineage development and tissue-specific immunity.
Molecular Dissection of Prethymic Progenitor Entry into the T Lymphocyte Developmental Pathway
Authors: Tydell C. Chace , David-Fung Elizabeth-Sharon , Moore Jonathan E. , Rowen Lee , Taghon Tom , Rothenberg Ellen V.
Published: April 2014

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-1 (TCF-1) (gene name Tcf7). To identify additional regulators of T cell specification, a cDNA library from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin−Sca-1+Kit+CD27−) and multipotent progenitors (Lin−Sca-1+Kit+CD27+), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bcl11b, TCF-1 (Tcf7), and HEBalt, Notch target Deltex1, Deltex3L, Fkbp5, Eva1, and Tmem131. Like GATA3 and Deltex1, Bcl11b, Fkbp5, and Eva1 were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only Bcl11b and HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative 1 and double negative 2) corresponding to T lineage specification. Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway.