bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine Low-Density Lipoprotein Receptor-Related Protein 1 (LRP) Is Required for Phagocytosis of Targets Bearing LRP Ligands but Is Not Required for C1q-Triggered Enhancement of Phagocytosis
Authors: Lillis Anna P. , Greenlee Mallary C. , Mikhailenko Irina , Pizzo Salvatore V. , Tenner Andrea J. , Strickland Dudley K. , Bohlson Suzanne S.
Published: April 2014

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.
Quantitative Regulation of B Cell Division Destiny by Signal Strength
Authors: Turner Marian L. , Hawkins Edwin D. , Hodgkin Philip D.
Published: April 2014

Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population’s division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.
Haemophilus influenzae Interacts with the Human Complement Inhibitor Factor H
Authors: Hallström Teresia , Zipfel Peter F. , Blom Anna M. , Lauer Nadine , Forsgren Arne , Riesbeck Kristian
Published: April 2014

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6–7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18–20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of ∼32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.
Dual Role of CD38 in Microglial Activation and Activation-Induced Cell Death
Authors: Mayo Lior , Jacob-Hirsch Jasmine , Amariglio Ninette , Rechavi Gideon , Moutin Marie-Jo , Lund Frances E. , Stein Reuven
Published: April 2014

Microglia, the resident immune cells of the CNS, are normally quiescent but become activated after infection or injury. Their properties then change, and they promote both repair and damage processes. The extent of microglial activation is regulated, in part, by activation-induced cell death (AICD). Although many apoptotic aspects of the microglial AICD mechanism have been elucidated, little is known about the connection between the activation step and the death process. Using mouse primary microglial cultures, we show that the ectoenzyme CD38, via its calcium-mobilizing metabolite cyclic-ADP-ribose (cADPR), helps promote microglial activation and AICD induced by LPS plus IFN-γ (LPS/IFN-γ), suggesting that CD38 links the two processes. Accordingly, CD38 expression and activity, as well as the intracellular calcium concentration ([Ca2+]i) in the primary microglia were increased by LPS/IFN-γ treatment. Moreover, CD38 deficiency or treatment with cADPR antagonists conferred partial resistance to LPS/IFN-γ-induced AICD and also reduced [Ca2+]i. Microglial activation, indicated by induced expression of NO synthase-2 mRNA and production of NO, secretion and mRNA expression of TNF-α and IL-12 p40, and expression of IL-6 mRNA, was attenuated by CD38 deficiency or cADPR-antagonist treatment. The observed effects of CD38 on microglial activation are probably mediated via a cADPR-dependent increase in [Ca2+]i and the effect on AICD by regulation of NO production. Our results thus suggest that CD38 significantly affects regulation of the amount and function of activated microglia, with important consequences for injury and repair processes in the brain.
Dendritic Cell Differentiation Induced by a Self-Peptide Derived from Apolipoprotein E
Authors: Stephens Tracey A. , Nikoopour Enayat , Rider Beverly J. , Leon-Ponte Matilde , Chau Thu A. , Mikolajczak Sebastian , Chaturvedi Pratibha , Lee-Chan Edwin , Flavell Richard A. , Haeryfar S. M. Mansour , Madrenas Joaquin , Singh Bhagirath
Published: April 2014

Dendritic cells (DCs) are professional APCs and potent stimulators of naive T cells. Since DCs have the ability to immunize or tolerize T cells they are unique candidates for use in immunotherapy. Our laboratory has discovered that a naturally processed self-peptide from apolipoprotein E, Ep1.B, induces DC-like morphology and surface marker expression in a murine monocytic cell line (PU5-1.8), human monocytic cell line (U937), murine splenocytes, and human peripheral blood monocytes. Microscopy and flow cytometric analysis revealed that Ep1.B-treated cells display decreased adherence to plastic and increased aggregation, dendritic processes, and expression of DC surface markers, including DEC-205, CD11c, B7.1, and B7.2. These effects were observed in both PU5-1.8 cells and splenocytes from various mouse strains including BALB/c, C57BL/6, NOD/Lt, and C3H/HeJ. Coadministration of Ep1.B with OVA antigenic peptide functions in dampening specific immune response to OVA. Ep1.B down-regulates proliferation of T cells and IFN-γ production and stimulates IL-10 secretion in immunized mice. Ep1.B-induced differentiation resulted in the activation of PI3K and MAPK signaling pathways, including ERK1/2, p38, and JNK. We also found that NF-κB, a transcription factor essential for DC differentiation, is critical in mediating the effects of Ep1.B. Ep1.B-induced differentiation is independent of MyD88-dependent pathway of TLR signaling. Cumulatively, these findings suggest that Ep1.B acts by initiating a signal transduction cascade in monocytes leading to their differentiation into DCs.
Differential Regulatory Function of Resting and Preactivated Allergen-Specific CD4+CD25+ Regulatory T Cells in Th2-Type Airway Inflammation
Authors: Saito Kanako , Torii Mie , Ma Ning , Tsuchiya Tomoko , Wang Linan , Hori Tomohide , Nagakubo Daisuke , Nitta Nao , Kanegasaki Shiro , Hieshima Kunio , Yoshie Osamu , Gabazza Esteban C. , Katayama Naoyuki , Shiku Hiroshi , Kuribayashi Kagemasa , Kato Takuma
Published: April 2014

Although CD4+CD25+ regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.
Human CD4+CD25+Foxp3+ Regulatory T Cells Do Not Constitutively Express IL-35
Authors: Bardel Emilie , Larousserie Frédérique , Charlot-Rabiega Pascaline , Coulomb-L'Herminé Aurore , Devergne Odile
Published: April 2014

EBV-induced gene 3 (EBI3) can associate with p28 to form the heterodimeric cytokine IL-27, or with the p35 subunit of IL-12 to form the EBI3/p35 heterodimer, recently named IL-35. In mice, IL-35 has been shown to be constitutively expressed by CD4+CD25+Foxp3+ regulatory T cells (Treg cells) and suggested to contribute to their suppressive activity. In this study, we investigated whether human Treg cells express IL-35. Double-staining analysis of human thymuses showed that neither Foxp3+ nor CD25+ cells coexpressed EBI3. Similarly, Foxp3+ cells present in human lymph nodes, tonsils, spleens, and intestines did not express EBI3. Consistent with these in situ observations, Treg cells purified from blood or tonsils were negative for EBI3 by immunoblotting. Other human T cell subsets, including effector T cells, naive and memory CD4+ T cells, CD8+ and γδ T cells also did not constitutively express EBI3, which contrasts with IL-35 expression observed in murine CD8+ and γδ T cells. Furthermore, although CD3/CD28 stimulation consistently induced low levels of EBI3 in various CD4+ T cell subsets, no EBI3 could be detected in CD3/CD28-stimulated Treg cells. RT-PCR analysis showed that, whereas p35 transcripts were detected in both Teff and Treg cells, EBI3 transcripts were detected only in activated Teff cells, but not in resting or activated Treg cells. Thus, in contrast to their murine counterpart, human Treg cells do not express detectable amounts of IL-35.
Thymocyte-Dendritic Cell Interactions near Sources of CCR7 Ligands in the Thymic Cortex
Authors: Ladi Ena , Schwickert Tanja A. , Chtanova Tatyana , Chen Ying , Herzmark Paul , Yin Xinye , Aaron Holly , Chan Shiao Wei , Lipp Martin , Roysam Badrinath , Robey Ellen A.
Published: April 2014

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals.
Phenotype Switching by Inflammation-Inducing Polarized Th17 Cells, but Not by Th1 Cells
Authors: Shi Guangpu , Cox Catherine A. , Vistica Barbara P. , Tan Cuiyan , Wawrousek Eric F. , Gery Igal
Published: April 2014

Th1 and Th17 cells are characterized by their expression of IFN-γ or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-γ suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-γ was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-γ or both IFN-γ and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-γ or IFN-γ/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20–25% nonpolarized cells than in recipients of 35–40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-γ or IFN-γ/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-γt was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-γ or IFN-γ/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.
IL-10 and TGF-β Redundantly Protect against Severe Liver Injury and Mortality during Acute Schistosomiasis
Authors: Herbert De'Broski R. , Orekov Tatyana , Perkins Charles , Finkelman Fred D.
Published: April 2014

The cytokines IL-10 and TGF-β regulate immunity and inflammation. IL-10 is known to suppress the extent of hepatic damage caused by parasite ova during natural infection with Schistosoma mansoni, but the role of TGF-β is less clear. Cytokine blockade studies in mice revealed that anti-IL-10R mAb treatment during acute infection modestly increased cytokine production and liver damage, whereas selective anti-TGF-β mAb treatment had marginal effects. In contrast, mice administered both mAbs developed severe hepatic inflammation, with enlarged, necrotic liver granulomas, cachexia, and >80% mortality by 8 wk postinfection, despite increased numbers of CD4+CD25+Foxp3+ T regulatory cells. Blocking both IL-10 and TGF-β at the onset of egg production also significantly increased IL-4, IL-6, TNF, IFN-γ, and IL-17 production and markedly increased hepatic, peritoneal, and splenic neutrophilia. In contrast, coadministration of anti-IL-10R and TGF-β mAbs had little effect upon parasite ova-induced intestinal pathology or development of alternatively activated macrophages, which are required to suppress intestinal pathology. This suggests that inflammation is controlled during acute S. mansoni infection by two distinct, organ-specific mechanisms: TGF-β and IL-10 redundantly suppress hepatic inflammation while intestinal inflammation is regulated by alternatively activated macrophages.
Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors
Authors: Sturm Eva M. , Schratl Petra , Schuligoi Rufina , Konya Viktoria , Sturm Gunter J. , Lippe Irmgard Th. , Peskar Bernhard A. , Heinemann Akos
Published: April 2014

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.
Pleural Mesothelial Cells Express Both BLT2 and PPARα and Mount an Integrated Response to Pleural Leukotriene B4
Authors: Pace Elisabetta , Ferraro Maria , Mody Christopher H. , Melis Mario R. , Scafidi Valeria , Bonanno Anna , Profita Mirella , Giarratano Antonino , Gjomarkaj Mark
Published: April 2014

Leukotriene B4 (LTB4) plays a crucial role in the recruitment of neutrophils into the pleural space. We identified for the first time the mechanisms by which LTB4 interacts with mesothelial cells and recruits neutrophils in the pleural compartment. Primary pleural mesothelial cells express both the proinflammatory receptor for LTB4 BLT2, and the anti-inflammatory receptor for LTB4, PPARα. Parapneumonic pleural effusions highly increase BLT2 expression and, via BLT2 activation, increase the adhesion between mesothelial cells and neutrophils and the expression of ICAM-1 on mesothelial cells. The block of PPARα further increases both cell adhesion and ICAM-1 expression. BLT2 activation promotes the activation, on mesothelial cells, of STAT-1 but not the activation of NF-κB transcription factor. The increase of ICAM-1 expression is achieved via increased tyrosine phosphorylation activity since herbimycin, a tyrosine kinase inhibitor, reduces and since Na orthovanadate, a tyrosine phosphatase inhibitor, further increases ICAM-1 expression. This study demonstrates that pleural mesothelial cells, expressing both proinflammatory and anti-inflammatory LTB4 receptors, are able to mount an integrated response to LTB4 with a prevalence of BLT2 activities in the presence of an inflammatory milieu within the pleura.
The Effector T Cells of Diabetic Subjects Are Resistant to Regulation via CD4+FOXP3+ Regulatory T Cells
Authors: Schneider Anya , Rieck Mary , Sanda Srinath , Pihoker Catherine , Greenbaum Carla , Buckner Jane H.
Published: April 2014

Defects in immune regulation have been implicated in the pathogenesis of diabetes in mouse and in man. In vitro assays using autologous regulatory (Treg) and responder effector (Teff) T cells have shown that suppression is impaired in diabetic subjects. In this study, we addressed whether the source of this defect is intrinsic to the Treg or Teff compartment of diabetic subjects. We first established that in type 1 diabetes (T1D) individuals, similar levels of impaired suppression were seen, irrespective of whether natural (nTreg) or adaptive Treg (aTreg) were present. Then using aTreg, we examined the ability of T1D aTreg to suppress Teff of healthy controls, as compared with the ability of control aTreg to suppress Teff of diabetic subjects. Taking this approach, we found that the aTregs from T1D subjects function normally in the presence of control Teff, and that the T1D Teff were resistant to suppression in the presence of control aTreg. This escape from regulation was seen with nTreg as well and was not transferred to control Teff coincubated with T1D Teff. Thus, the “defective regulation” in T1D is predominantly due to the resistance of responding T cells to Treg and is a characteristic intrinsic to the T1D Teff. This has implications with respect to pathogenic mechanisms, which underlie the development of disease and the target of therapies for T1D.
Breaking of CD8+ T Cell Tolerance through In Vivo Ligation of CD40 Results in Inhibition of Chronic Graft-versus-Host Disease and Complete Donor Cell Engraftment
Authors: Kim Juyang , Park Keunhee , Kim Hyun J. , Kim Jiyoung , Kim Hyun-A , Jung Daehee , Kim Hye J. , Choi Hye-Jeong , Choi Suck-Young , Seo Kwang W. , Cho Hong R. , Kwon Byungsuk
Published: April 2014

In the DBA/2 → unirradiated (C57BL/6 × DBA/2)F1 model of chronic graft-vs-host disease (cGVHD), donor CD4+ T cells play a critical role in breaking host B cell tolerance, while donor CD8+ T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8+ T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8+ T cell anergy such that subsequently activated donor CD8+ T cells deleted host CD4+ T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8+ T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8+ T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.
Telomerase-Based Pharmacologic Enhancement of Antiviral Function of Human CD8+ T Lymphocytes
Authors: Fauce Steven Russell , Jamieson Beth D. , Chin Allison C. , Mitsuyasu Ronald T. , Parish Stan T. , Ng Hwee L. , Ramirez Kitchen Christina M. , Yang Otto O. , Harley Calvin B. , Effros Rita B.
Published: April 2014

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8+ CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8+ T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.
Uncoupling of Induced Protein Processing from Maturation in Dendritic Cells Exposed to a Highly Antigenic Preparation from a Helminth Parasite
Authors: Marshall Fraser A. , Pearce Edward J.
Published: April 2014

TLR ligands induce dendritic cell (DC) maturation. During this process, cells initiate proteolytic degradation of internalized protein Ags into peptides that complex with MHC class II (MHC II) and simultaneously increase expression of costimulatory molecules and of cytokines such as IL-6, IL-12, and IL-23. In these ways, TLR-activated DCs are able to activate naive Th cells and initiate Th1 and Th17 responses, and TLR ligands thus serve as adjuvants for these types of responses. In contrast, products from helminth parasites generally do not activate DCs and act as adjuvants for Th2 response induction. We have explored the underlying basis for this form of adjuvanticity. We show that exposure of DCs to soluble Ags from the eggs of the helminth parasite Schistosoma mansoni (schistosome egg Ag (SEA)) leads to the induction of proteolysis of internalized Ag. This occurs in the absence of significant induction of costimulatory molecule expression or production of proinflammatory cytokines. SEA-induced Ag processing occurs independently of MyD88 or Toll/IL-1 receptor domain containing adaptor inducing IFN-β (Trif), but is significantly attenuated by inhibition of p38, but not ERK, signaling. In DCs exposed to SEA, ligation of CD40 provides a necessary second signal that stimulates costimulatory molecule expression, allowing DCs to mature into capable APCs. Collectively, the data demonstrate the existence of a MyD88/Trif-independent, p38-dependent pathway of Ag processing in DCs, which is uncoupled from conventional DC maturation and is associated with induction of Th2-type immune responses.
Protective Immunity to Systemic Infection with AttenuatedSalmonella entericaserovar Enteritidis in the Absence of IL-12 Is Associated with IL-23-Dependent IL-22, but Not IL-17
Authors: Schulz Silke M. , Köhler Gabriele , Schütze Nicole , Knauer Jens , Straubinger Reinhard K. , Chackerian Alissa A. , Witte Ellen , Wolk Kerstin , Sabat Robert , Iwakura Yoichiro , Holscher Christoph , Müller Uwe , Kastelein Robert A. , Alber Gottfried
Published: April 2014

IL-12 is essential for protective T cell-mediated immunity against Salmonella infection. To characterize the role of the related cytokine IL-23, wild-type (WT) C57BL/6 and p19−/− mice were infected systemically with an attenuated strain of Salmonella enterica serovar Enteritidis (S. Enteritidis). IL-23-deficient mice controlled infection with S. Enteritidis similarly as WT mice. Similar IFN-γ production as compared with WT mice, but defective IL-17A and IL-22 production was found in the absence of IL-23. Nevertheless, although IL-23 is required for T cell-dependent cytokine responses, IL-23 is dispensable for protection against S. Enteritidis when IL-12 is present. To analyze the role of IL-23 in the absence of IL-12, low doses of S. Enteritidis were administered to p35−/− mice (lacking IL-12), p35/19−/− mice (lacking IL-12 and IL-23), p35/40−/− mice (lacking IL-12, IL-23, and homodimeric IL-12p40), or p35/IL-17A−/− mice (lacking IL-12 and IL-17A). We found survival of p35−/− and p35/IL-17A−/− mice, whereas p35/19−/− and p35/40−/− mice died within 3–6 wk and developed liver necrosis. This indicates that IL-23, but not homodimeric IL-12p40, is required for protection, which, surprisingly, is independent of IL-17A. Moreover, protection was associated with IL-22, but not IL-17F or IL-21 expression or with neutrophil recruitment. Finally, anti-IL-22 treatment of S. Enteritidis-infected p35−/− mice resulted in liver necrosis, indicating a central role of IL-22 in hepatocyte protection during salmonellosis. In conclusion, IL-23-dependent IL-22, but not IL-17 production is associated with protection against systemic infection with S. Enteritidis in the absence of IL-12.
SOCS1 Regulates the IFN but Not NFκB Pathway in TLR-Stimulated Human Monocytes and Macrophages
Authors: Prêle Cecilia M. , Woodward Eleanor A. , Bisley Jacqueline , Keith-Magee April , Nicholson Sandra E. , Hart Prue H.
Published: April 2014

SOCS1 can regulate TLR-mediated signal transduction, yet mechanistic studies in murine macrophages have been confusing and contradictory. This study has used an adenoviral transfection system to determine the role of SOCS1 in the regulation of TNF-α production by activated human monocytes. Monocytes were infected with AdV-SOCS1 or with an empty vector control, AdV-GFP, for 24 h before activation with the TLR4 ligand, LPS. SOCS1 did not regulate TNF-α mRNA or protein production within the first two hours of TLR4 activation. However, SOCS1 suppressed the sustained production of TNF-α by primary human monocytes and synovial fluid macrophages ex vivo. In addition, SOCS1 regulated the production of IL-6, but not IL-10, by monocytes. Analysis of the early signaling pathway downstream of TLR4 demonstrated that SOCS1 had no regulatory effect on the activation or on the DNA binding capacity of NFκB. The late effects of LPS are mediated in part through the MyD88-independent pathway activating IRF3 and initiating the production of IFN-β. In response to adenoviral infection and before LPS exposure, monocytes expressed enhanced levels of IFN-β and Myxovirus A mRNA, an anti-viral molecule characterizing IFN-β activity. These two genes were reduced in AdV-SOCS1-infected cells. Further, SOCS1 regulated IFN-dependent pathways in LPS-activated cells as evidenced by reduced IFN-β production and STAT1 phosphorylation. Using AdV-infection to dissect SOCS1 control of IFN-dependent pathways, this study suggests that SOCS1-regulation of the IFN-dependent component of the LPS-induced TLR4 signaling pathway may contribute to the down-regulation of inflammatory cytokine production by AdV-SOCS1-infected human monocytes.
Sulfatide, A Major Lipid Component of Myelin Sheath, Activates Inflammatory Responses As an Endogenous Stimulator in Brain-Resident Immune Cells
Authors: Jeon Sae-Bom , Yoon Hee Jung , Park Se-Ho , Kim In-Hoo , Park Eun Jung
Published: April 2014

Sulfatide, a major lipid component of myelin sheath, participates in diverse cellular events of the CNS, and its cellular level has recently been implicated in many inflammation-associated neuronal diseases. Herein, we report that sulfatide alone can trigger pathological inflammatory responses in glia, brain-resident immune cells. We show that sulfatide changed the morphology of primary microglia to their activated form, and it significantly induced the production of various inflammatory mediators in primary microglia and astrocytes. Moreover, sulfatide rapidly triggered the phosphorylation of p38, ERK, and JNK within 30 min, and it markedly enhanced the NF binding activity to NF-κB and AP-1 binding elements. However, nonsulfated galactocerebroside, another major lipid component of myelin, had no effect on activation of glia. We further reveal that CD1d did not contribute to sulfatide-stimulated activation of MAPKs, although its expression was enhanced by sulfatide and sulfatide-treated microglial cells actually stimulated type II NKT cells. Sulfatide significantly stimulated the phosphorylation of MAPKs in glia from CD1d-deficient mice, and the phosphorylation levels were similar to those in wild-type littermates. Sulfatide-triggered inflammatory events appear to occur at least in part through an L-selectin-dependent mechanism. L-selectin was dramatically down-regulated upon exposure to sulfatide, and inhibition of L-selectin resulted in suppression of sulfatide-triggered responses. Collectively, these results show that abnormally released sulfatide at demyelinated regions may act as an endogenous stimulator in the brain immune system, thus causing and further exacerbating pathological conditions in the brain.
Intrarectal Vaccination with Recombinant Vaccinia Virus Expressing Carcinoembronic Antigen Induces Mucosal and Systemic Immunity and Prevents Progression of Colorectal Cancer
Authors: Kim-Schulze Seunghee , Kim Hong Sung , Wainstein Alberto , Kim Dae Won , Yang Wein Cui , Moroziewicz Dorota , Mong Phyllus Y. , Bereta Michal , Taback Bret , Wang Qin , Kaufman Howard L.
Published: April 2014

The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8+ CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4+ and CD8+ T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.
Detection of Complement Activation on Antigen Microarrays Generates Functional Antibody Profiles and Helps Characterization of Disease-Associated Changes of the Antibody Repertoire
Authors: Papp Krisztián , Végh Péter , Miklós Kata , Németh Julianna , Rásky Klára , Péterfy Ferenc , Erdei Anna , Prechl József
Published: April 2014

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response. To generate functional Ab profiles we simultaneously tested not only IgM, IgG, and IgA binding to, but also complement activation by, a panel of endogenous and exogenous Ags printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular Ag in a given individual cannot be predicted by the measurement of Ag-specific Abs, despite a general correlation between the amount of Ag-bound Ab and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an Ag and individual variations for a given isotype, resulting in altered complement activation potential. Thus, Ag-specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of Ags, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and Ab binding. Since cleavage fragments of C3 mediate important immunological processes, we propose that measurement of their deposition on Ag microarrays, in addition to Ab profiling, can provide useful functional signature about the tested serum.
Regulated Movement of CD4 In and Out of the Immunological Synapse
Authors: Kao Henry , Lin Joseph , Littman Dan R. , Shaw Andrey S. , Allen Paul M.
Published: April 2014

The mechanism underlying the transient accumulation of CD4 at the immunological synapse (IS) and its significance for T cell activation are not understood. To investigate these issues, we mutated a serine phosphorylation site (S408) in the cytoplasmic tail of murine CD4. Preventing phosphorylation of S408 did not block CD4 recruitment to the IS; rather, it blocked the ability of CD4 to leave the IS. Surprisingly, enhanced and prolonged CD4 accumulation at the supramolecular activation cluster in the contact area had no functional consequence for T cell activation, cytokine production, or proliferation. Protein kinase C θ (PKCθ)-deficient T cells also displayed enhanced and prolonged accumulation of wild-type CD4 at the IS, indicating that θ is the critical PKC isoform involved in CD4 movement. These findings suggest a model wherein recruitment of CD4 to the IS allows its phosphorylation by PKCθ and subsequent removal from the IS. Thus, an important role for PKCθ in T cell activation involves its recruitment to the IS, where it phosphorylates specific substrates that help to maintain the dynamism of protein turnover at the IS.
B Lymphocytes Promote Expansion of Regulatory T Cells in Oral Tolerance: Powerful Induction by Antigen Coupled to Cholera Toxin B Subunit
Authors: Sun Jia-Bin , Flach Carl-Fredrik , Czerkinsky Cecil , Holmgren Jan
Published: April 2014

Mucosal administration of Ag conjugated to cholera toxin B subunit (CTB) can efficiently induce peripheral immunologic tolerance, so-called oral tolerance, associated with development of Foxp3+CD25+CD4+ regulatory T (Treg) cells. Using an established sublingual tolerization regimen with Ag(OVA)/CTB conjugate, wherein CTB mediates Ag uptake and presentation by most B lymphocytes irrespective of their Ag specificity, we have assessed the importance of B cells for induction of Ag-specific Treg cells and oral tolerance. We found that Treg cells are reduced in μMT−/− B cell-deficient mice compared with wild-type (WT) mice. After sublingual Ag/CTB treatment, Treg cells increased much more in WT than in μMT−/− mice; however, adoptive transfer of B cells before treatment normalized Treg cell development and functional oral tolerance. B cells from OVA/CTB-treated mice expressed more IL-10 and less CD86 than control B cells. Adoptive transfer of these cells before parenteral immunization with OVA led to efficient suppression of proliferation and to induction of apoptotic depletion of Ag-specific CD25−CD4+ effector T cells associated with the expansion of Treg cells. However, also OVA/CTB-treated μMT−/− mice could suppress the immune response to parenteral immunization with OVA, which was associated with a strong increase in Foxp3−CD4+ T cells expressing LAP/TGF-β. Our results indicate that mucosal tolerance comprises at least two separate pathways: one being B cell dependent and associated with expansion of Treg cells and Treg-mediated suppression and depletion of effector T cells, and one being B cell independent and associated with development of Foxp3−LAP+TGF-β+ regulatory T cells.
IP3 Receptor-Mediated Ca2+ Release in Naive CD4 T Cells Dictates Their Cytokine Program
Authors: Nagaleekar Viswas K. , Diehl Sean A. , Juncadella Ignacio , Charland Colette , Muthusamy Natarajan , Eaton Sheri , Haynes Laura , Garrett-Sinha Lee Ann , Anguita Juan , Rincón Mercedes
Published: April 2014

IP3 (inositol 1,4,5-trisphosphate) receptors (IP3Rs) regulate the release of Ca2+ from intracellular stores in response to IP3. Little is known about regulation of the expression of IP3Rs and their role during the activation of CD4 T cells. In this study we show that mouse naive CD4 T cells express IP3R1, IP3R2, and IP3R3, but that gene expression of IP3R3 primarily is down-regulated upon activation due to loss of the Ets-1 transcription factor. Down-regulation of IP3R expression in activated CD4 T cells is associated with the failure of TCR ligation to trigger Ca2+ release in these cells. We also show that down-regulation of specific IP3Rs in activated CD4 T cells correlates with the requirement of IP3R-mediated Ca2+ release only for the induction of, but not for the maintenance of, IL-2 and IFN-γ expression. Interestingly, while inhibition of IP3R function early during activation blocks IL-2 and IFN-γ production, it promotes the production of IL-17 by CD4 T cells. Thus, IP3Rs play a key role in the activation and differentiation of CD4 T cells. The immunosuppressive effect of pharmacological blockers of these receptors may be complicated by promoting the development of inflammatory CD4 T cells.
T-Bet Dependent Removal of Sin3A-Histone Deacetylase Complexes at the Ifng Locus Drives Th1 Differentiation
Authors: Chang Shaojing , Collins Patrick L. , Aune Thomas M.
Published: April 2014

Forming and removing epigenetic histone marks at gene loci are central processes in differentiation. Here, we explored mechanisms establishing long-range H4 acetylation marks at the Ifng locus during Th1 lineage commitment. In Th0 cells, histone deacetylase (HDAC)-Sin3A complexes recruited to the Ifng locus actively prevented accumulation of H4 acetylation marks. Th1 differentiation caused loss of HDAC-Sin3A complexes by T-bet-dependent mechanisms and accumulation of H4 acetylation marks. HDAC-Sin3A complexes were absent from the locus in NOD Th0 cells, obviating the need for Th1 differentiation signals to establish histone marks and Th1 differentiation. Thus, Ifng transcription is actively prevented in Th0 cells via epigenetic mechanisms and epigenetic defects allow unregulated Ifng transcription that may contribute to autoimmunity.
IL-17A Enhances Vitamin D3-Induced Expression of Cathelicidin Antimicrobial Peptide in Human Keratinocytes
Authors: Peric Mark , Koglin Sarah , Kim Song-Min , Morizane Shin , Besch Robert , Prinz Jörg C. , Ruzicka Thomas , Gallo Richard L. , Schauber Jürgen
Published: April 2014

Cathelicidin is strongly expressed in lesional skin in psoriasis and may play an important role as both an antimicrobial peptide and as an autoinflammatory mediator in this chronic skin disease. The mechanism of increased cathelicidin in psoriatic keratinocytes is not known, but recent observations have found that psoriasis has abundant Th17 cells that produce IL-17A and IL-22. We found that human keratinocytes stimulated with supernatants from T cells isolated from lesional psoriatic skin increased expression of cathelicidin when stimulated in the presence of 1,25-dihydroxyvitamin D3 (1,25D3). This increase was signaled through the IL-17RA. In vitro, IL-17A, but not IL-22, enhanced cathelicidin mRNA and peptide expression in keratinocytes dependent on the presence of 1,25D3. At the same time, coincubation with 1,25D3 blocked induction of human β-defensin 2 (HBD2), IL-6, and IL-8, which are other target genes of IL-17A. Act1, an adaptor associated with IL-17RA and essential for IL-17A signaling, mediated cathelicidin induction, as its suppression by small interfering RNA inhibited HBD2 and cathelicidin. Both, 1,25D3 and IL-17A signaled cathelicidin induction through MEK-ERK. These results suggest that increased IL-17A in psoriatic skin increases cathelicidin through a vitamin D3-, Act1-, and MEK-ERK-dependent mechanism. Therapy targeting this cathelicidin-regulating system might be beneficial in patients suffering from psoriasis.
Lipocalin 2-Dependent Inhibition of Mycobacterial Growth in Alveolar Epithelium
Authors: Saiga Hiroyuki , Nishimura Junichi , Kuwata Hirotaka , Okuyama Megumi , Matsumoto Sohkichi , Sato Shintaro , Matsumoto Makoto , Akira Shizuo , Yoshikai Yasunobu , Honda Kenya , Yamamoto Masahiro , Takeda Kiyoshi
Published: April 2014

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.