bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Wiskott-Aldrich Syndrome Protein in Dendritic Cells Regulates Synapse Formation and Activation of Naive CD8+ T Cells
Authors: Pulecio Julian , Tagliani Elisa , Scholer Alix , Prete Francesca , Fetler Luc , Burrone Oscar R. , Benvenuti Federica
Published: April 2014

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polimerization in hematopoietic cells. Mutations in WASp cause a severe immunodeficiency characterized by defective initiation of primary immune response and autoimmunity. The contribution of altered dendritic cells (DCs) functions to the disease pathogenesis has not been fully elucidated. In this study, we show that conventional DCs develop normally in WASp-deficient mice. However, Ag targeting to lymphoid organ-resident DCs via anti-DEC205 results in impaired naive CD8+ T cell activation, especially at low Ag doses. Altered trafficking of Ag-bearing DCs to lymph nodes (LNs) accounts only partially for defective priming because correction of DCs migration does not rescue T cell activation. In vitro and in vivo imaging of DC-T cell interactions in LNs showed that cytoskeletal alterations in WASp null DCs causes a reduction in the ability to form and stabilize conjugates with naive CD8+ T lymphocytes both in vitro and in vivo. These data indicate that WASp expression in DCs regulates both the ability to traffic to secondary lymphoid organs and to activate naive T cells in LNs.
Enterocyte-Derived TAK1 Signaling Prevents Epithelium Apoptosis and the Development of Ileitis and Colitis
Authors: Kajino-Sakamoto Rie , Inagaki Maiko , Lippert Elisabeth , Akira Shizuo , Robine Sylvie , Matsumoto Kunihiro , Jobin Christian , Ninomiya-Tsuji Jun
Published: April 2014

Recent studies have revealed that TAK1 kinase is an essential intermediate in several innate immune signaling pathways. In this study, we investigated the role of TAK1 signaling in maintaining intestinal homeostasis by generating enterocyte-specific constitutive and inducible gene-deleted TAK1 mice. We found that enterocyte-specific constitutive TAK1-deleted mice spontaneously developed intestinal inflammation as observed by histological analysis and enhanced expression of IL-1β, MIP-2, and IL-6 around the time of birth, which was accompanied by significant enterocyte apoptosis. When TAK1 was deleted in the intestinal epithelium of 4-wk-old mice using an inducible knockout system, enterocytes underwent apoptosis and intestinal inflammation developed within 2–3 days following the initiation of gene deletion. We found that enterocyte apoptosis and intestinal inflammation were strongly attenuated when enterocyte-specific constitutive TAK1-deleted mice were crossed to TNF receptor 1−/− mice. However, these mice later (>14 days) developed ileitis and colitis. Thus, TAK1 signaling in enterocytes is essential for preventing TNF-dependent epithelium apoptosis and the TNF-independent development of ileitis and colitis. We propose that aberration in TAK1 signaling might disrupt intestinal homeostasis and favor the development of inflammatory disease.
B Cells Induce Tolerance by Presenting Endogenous Peptide-IgG on MHC Class II Molecules via an IFN-γ-Inducible Lysosomal Thiol Reductase-Dependent Pathway
Authors: Su Yan , Carey Gregory , Marić Maja , Scott David W.
Published: April 2014

We have previously demonstrated that splenic B cells, transduced with peptide-IgG fusion proteins, are efficient tolerogenic APCs in vivo. Specific hyporesponsiveness to epitopes encoded in the peptide-IgG fusion protein has been achieved to over one dozen Ags, and clinical efficacy has been established in animal models for several autoimmune diseases and hemophilia. Previous studies also demonstrated that tolerance in this system requires MHC class II expression by the transduced B cells. Yet, the mechanisms of this B cell tolerogenic processing pathway remain unclear. In this study, we show that MHC class II molecules on tolerogenic B cells present epitopes derived from endogenous, but not exogenous (secreted), peptide-IgG fusion protein. These class II epitopes from the IgG fusion protein are processed in lysosomes/endosomes in an IFN-γ-inducible lysosomal thiol reductase-dependent manner. We suggest that the MHC class II presentation of endogenously produced fusion protein epitopes represents a novel mechanism for tolerance induced by peptide-IgG-transduced B cells. An understanding of this process might provide insights into central and peripheral tolerance induced by other professional and nonprofessional APCs.
A Novel Virus Carrier State to Evaluate Immunotherapeutic Regimens: Regulatory T Cells Modulate the Pathogenicity of Antiviral Memory Cells
Authors: Truong Phi , McGavern Dorian B.
Published: April 2014

Restrictions in the diversity of an adaptive immune repertoire can facilitate viral persistence. Because a host afflicted with an immune deficiency is not likely to purge a persistent infection using endogenous mechanisms, it is important to explore adoptive therapies to supplement the host with a functional immune defense. In this study, we describe a virus carrier state that results from introducing lymphocytic choriomeningitis virus (LCMV) into adult mice possessing a restricted T cell repertoire. On infection of these mice, LCMV establishes systemic persistence, and within the CNS the virus infects astrocytes (and later oligodendrocytes) rather than its traditional parenchymal target neurons. To determine whether LCMV could be purged from a novel target selection in the absence of an endogenous immune repertoire, we adoptively transferred virus-specific memory cells into adult carrier mice. The memory cells purged virus from the periphery as well as the CNS, but they induced fatalities not typically associated with adoptive immunotherapy. When the repertoire of the recipient mice was examined, a deficiency in natural regulatory T cells was noted. We therefore supplemented carrier mice with regulatory T cells and simultaneously performed adoptive immunotherapy. Cotransfer of regulatory T cells significantly reduced mortality while still permitting the antiviral memory cells to purge the persistent infection. These data indicate that regulatory T cells can be used therapeutically to lessen the pathogenicity of virus-specific immune cells in an immunodeficient host. We also propose that the novel carrier state described herein will facilitate the study of immunotherapeutic regimens.
Leukotriene B4 Receptor 1 Expression on Dendritic Cells Is Required for the Development of Th2 Responses and Allergen-Induced Airway Hyperresponsiveness
Authors: Miyahara Nobuaki , Ohnishi Hiroshi , Matsuda Hiroyuki , Miyahara Satoko , Takeda Katsuyuki , Koya Toshiyuki , Matsubara Shigeki , Okamoto Masakazu , Dakhama Azzeddine , Haribabu Bodduluri , Gelfand Erwin W.
Published: April 2014

Dendritic cells (DC) are important APCs that control allergen-induced airway responses by interacting directly with T cells. Leukotriene B4 (LTB4), interacting with its high-affinity receptor, LTB4 receptor 1 (BLT1), is known to attract and activate leukocytes during inflammation. We have previously shown that BLT1 expression on Ag-primed T cells is required for the development of airway hyperresponsiveness (AHR; Miyahara et al. 2005. Am. J. Respir. Crit. Care Med. 172: 161–167). However, the role for the LTB4-BLT1 pathway in DC function in allergen-induced airway responses has not been defined. Bone marrow-derived DCs (BMDC) were generated. Naive BALB/c mice received OVA-pulsed BLT1-deficient (BLT1−/−) BMDCs or wild-type BMDCs intratracheally and were then challenged with OVA for 3 days. Airway responses were monitored 48 h after the last allergen challenge. BLT1−/− BMDCs showed normal maturation judged from surface expression of CD markers. Compared with recipients of wild-type BMDCs, mice that received BLT1−/− BMDCs developed significantly lower AHR to inhaled methacholine, lower goblet cell metaplasia, and eosinophilic infiltration in the airways and decreased levels of Th2 type cytokines in the bronchoalveolar lavage fluid. Migration of BLT1−/− BMDCs into peribronchial lymph nodes was significantly impaired compared with BLT1+/+ BMDCs after intratracheal instillation. These data suggest that BLT1 expression on DCs is required for migration of DCs to regional lymph nodes as well as in the development of AHR and airway inflammation.
Peripheral T Lymphocytes Recirculating Back into the Thymus Can Mediate Thymocyte Positive Selection
Authors: Kirberg Jörg , Bosco Nabil , Deloulme Jean-Christophe , Ceredig Rod , Agenès Fabien
Published: April 2014

The thymus continuously produces T lymphocytes that contribute to the maintenance of the peripheral T cell pool. Since peripheral recirculating T cells represent a very minor population among total thymocytes in normal animals, the relationship between the thymus and secondary lymphoid organs is generally considered unidirectional. Recently, several reports have described the presence of recirculating T cells in the thymus, raising issues regarding their possible function. In this article, we show that the niche for recirculating T cells in the thymus, i.e., their absolute number, is the same in lymphopenic and normal mice. Using a novel combination of TCR-transgenic mice in which the ligand necessary for positive selection of host T cells is only expressed by transferred donor T cells, we show that mature T cells recirculating back to the thymus can mediate positive selection.
Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/Pulmonary and Activation-Regulated Chemokine Induction
Authors: Li Xuehua , Syrovets Tatiana , Paskas Svetlana , Laumonnier Yves , Simmet Thomas
Published: April 2014

Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-α or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-α-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83+ DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.
Osteal Tissue Macrophages Are Intercalated throughout Human and Mouse Bone Lining Tissues and Regulate Osteoblast Function In Vitro and In Vivo
Authors: Chang Ming K. , Raggatt Liza-Jane , Alexander Kylie A. , Kuliwaba Julia S. , Fazzalari Nicola L. , Schroder Kate , Maylin Erin R. , Ripoll Vera M. , Hume David A. , Pettit Allison R.
Published: April 2014

Resident macrophages are an integral component of many tissues and are important in homeostasis and repair. This study examines the contribution of resident tissue macrophages to bone physiology. Using immunohistochemistry, we showed that a discrete population of resident macrophages, OsteoMacs, was intercalated throughout murine and human osteal tissues. OsteoMacs were distributed among other bone lining cells within both endosteum and periosteum. Furthermore, OsteoMacs were coisolated with osteoblasts in murine bone explant and calvarial preparations. OsteoMacs made up 15.9% of calvarial preparations and persisted throughout standard osteoblast differentiation cultures. Contrary to previous studies, we showed that it was OsteoMacs and not osteoblasts within these preparations that responded to pathophysiological concentrations of LPS by secreting TNF. Removal of OsteoMacs from calvarial cultures significantly decreased osteocalcin mRNA induction and osteoblast mineralization in vitro. In a Transwell coculture system of enriched osteoblasts and macrophages, we demonstrated that macrophages were required for efficient osteoblast mineralization in response to the physiological remodeling stimulus, elevated extracellular calcium. Notably, OsteoMacs were closely associated with areas of bone modeling in situ, forming a distinctive canopy structure covering >75% of mature osteoblasts on diaphyseal endosteal surfaces in young growing mice. Depletion of OsteoMacs in vivo using the macrophage-Fas-induced apoptosis (MAFIA) mouse caused complete loss of osteoblast bone-forming surface at this modeling site. Overall, we have demonstrated that OsteoMacs are an integral component of bone tissues and play a novel role in bone homeostasis through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics.
Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa
Authors: Walsh Catherine , Gangloff Monique , Monie Tom , Smyth Tomoko , Wei Bin , McKinley Trevelyan J. , Maskell Duncan , Gay Nicholas , Bryant Clare
Published: April 2014

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57–66 and 82–89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071–1082) of the ligand-bound TLR1/2 ectodomain heterodimer.
Lambda Light Chain Revision in the Human Intestinal IgA Response
Authors: Su Wen , Gordon John N. , Barone Francesca , Boursier Laurent , Turnbull Wayne , Mendis Surangi , Dunn-Walters Deborah K. , Spencer Jo
Published: April 2014

Revision of Ab L chains by secondary rearrangement in mature B cells has the potential to change the specific target of the immune response. In this study, we show for the first time that L chain revision is normal and widespread in the largest Ab producing population in man: intestinal IgA plasma cells (PC). Biases in the productive and non-productive repertoire of λ L chains, identification of the circular products of rearrangement that have the characteristic biases of revision, and identification of RAG genes and protein all reflect revision during normal intestinal IgA PC development. We saw no evidence of IgH revision, probably due to inappropriately orientated recombination signal sequences, and little evidence of κ-chain revision, probably due to locus inactivation by the κ-deleting element. We propose that the λ L chain locus is available and a principal modifier and diversifier of Ab specificity in intestinal IgA PCs.
Biphasic Regulation of Il2 Transcription in CD4+ T Cells: Roles for TNF-α Receptor Signaling and Chromatin Structure
Authors: McKarns Susan C. , Schwartz Ronald H.
Published: April 2014

We describe a novel biphasic regulation of Il2 transcription in naive CD4+ T cells. Few (∼5%) CD4+ T cells transcribe Il2 within 6 h of anti-TCR-β plus anti-CD28 stimulation (early phase). Most naive CD4+ T cells do not initiate Il2 transcription until after an additional ∼12 h of T cell stimulation (late phase). In comparison, essentially all previously activated (Pre-Ac) CD4+ T cells that transcribe Il2 do so with an early-phase response. Late-phase Il2 expression mostly requires c-Rel, CD28, and TNFR signaling. In contrast, early-phase transcription is only partly c-Rel and CD28 dependent and TNFR independent. There was also increased stable DNA accessibility at the Il2 locus and elevated c-Rel expression in resting Pre-Ac CD4+ cells. Upon T cell activation, a faster and greater increase in DNA accessibility as well as c-Rel nuclear expression were observed in Pre-Ac CD4+ cells relative to naive CD4+ T cells. In addition, both acetylated histone H3 and total H3 decreased at the Il2 locus upon rechallenge of Pre-Ac CD4+ T cells, whereas increased acetylated histone H3 with no change in total H3 was observed following activation of naive CD4+ T cells. We propose a model in which nucleosome disassembly facilitates rapid initiation of Il2 transcription in CD4+ T cells, and suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4+ T cells. This is provided, at least partially, by TNFR signaling during priming, but not during recall.
Two Loci on Chromosome 15 Control Experimentally Induced Arthritis through the Differential Regulation of IL-6 and Lymphocyte Proliferation
Authors: Glant Tibor T. , Szántó Sándor , Vegvari Aniko , Szabo Zoltan , Kis-Toth Katalin , Mikecz Katalin , Adarichev Vyacheslav A.
Published: April 2014

Using genetic linkage analysis of proteoglycan-induced arthritis (PGIA), a murine model for rheumatoid arthritis, we identified two loci, Pgia8 and Pgia9, on chromosome 15 (chr15) that appear to be implicated in disease susceptibility. Immunization of congenic strains carrying the entire chr15 and separately each of the two loci of DBA/2 arthritis-resistant origin in susceptible BALB/c background confirmed locations of two loci on chr15: the major Pgia9 and lesser Pgia8 locus. Distal part of chr15 (Pgia9) showed a major suppressive effect on PGIA susceptibility in females (40%, p < 0.001), whereas the effect of this locus in congenic males was still significant but weaker. Proximal part of chr15 (Pgia8) demonstrated mild and transient effect upon arthritis; this effect was PGIA-promoting in males and suppressive in females. Pgia8 and Pgia9 loci demonstrated an additive mode of inheritance, since when they were both incorporated in consomic chr15 strain, the total effect was a sum of the two loci. Using F2 population of the intercross of wild-type and chr15 consomic strain, we confirmed and refined quantitative trait locus positions and identified a strong correlation between disease susceptibility and lymphocyte-producing cytokines of TNF-α and IL-6. Both Pgia8 and Pgia9 loci on chr15 appear to control IL-6 production in spleen cultures of arthritic mice, providing an important link to the mechanism of autoimmune inflammation.
Balancing Selection Is the Main Force Shaping the Evolution of Innate Immunity Genes
Authors: Ferrer-Admetlla Anna , Bosch Elena , Sikora Martin , Marquès-Bonet Tomàs , Ramírez-Soriano Anna , Muntasell Aura , Navarro Arcadi , Lazarus Ross , Calafell Francesc , Bertranpetit Jaume , Casals Ferran
Published: April 2014

The evolutionarily recent geographic expansion of humans, and the even more recent development of large, relatively dense human settlements, has exposed our species to new pathogenic environments. Potentially lethal pathogens are likely to have exerted important selective pressures on our genome, so immunity genes can be expected to show molecular signatures of the adaptation of human populations to these recent conditions. While genes related to the acquired immunity system have indeed been reported to show traces of local adaptation, little is known about the response of the innate immunity system. In this study, we analyze the variability patterns in different human populations of fifteen genes related to innate immunity. We have used both single nucleotide polymorphism and sequence data, and through the analysis of interpopulation differentiation, the linkage disequilibrium pattern, and intrapopulation diversity, we have discovered some signatures of positive and especially balancing selection in these genes, thus confirming the importance of the immune system genetic plasticity in the evolutionary adaptive process. Interestingly, the strongest evidence is found in three TLR genes and CD14. These innate immunity genes play a pivotal role, being involved in the primary recognition of pathogens. In general, more evidences of selection appear in the European populations, in some case possibly related to severe population specific pressures. However, we also describe evidence from African populations, which may reflect parallel or long-term selective forces acting in different geographic areas.
IFN-γ Regulated Chemokine Production Determines the Outcome of Staphylococcus aureus Infection
Authors: McLoughlin Rachel M. , Lee Jean C. , Kasper Dennis L. , Tzianabos Arthur O.
Published: April 2014

Immunomodulatory therapy represents an attractive approach in treating multidrug-resistant infections. Developing this therapy necessitates a lucid understanding of host defense mechanisms. Neutrophils represent the first line of systemic defense during Staphylococcus aureus infections. However, recent research suggests that survival of S. aureus inside neutrophils may actually contribute to pathogenesis, indicating that neutrophil trafficking to the infection site must be tightly regulated to ensure efficient microbial clearance. We demonstrate that neutrophil-regulating T cells are activated during S. aureus infection and produce cytokines that control the local neutrophil response. S. aureus capsular polysaccharide activates T cell production of IFN-γ in a novel MHC class II-dependent mechanism. During S. aureus surgical wound infection, the presence of IFN-γ at the infection site depends upon αβTCR+ cells and functions to regulate CXC chemokine production and neutrophil recruitment in vivo. We note that the reduced neutrophil response seen in IFN-γ−/− mice during S. aureus infection is associated with reduced tissue bacterial burden. CXC chemokine administration to the infection site resulted in an increased survival of viable S. aureus inside neutrophils isolated from the wound. These data demonstrate that T cell-derived IFN-γ generates a neutrophil-rich environment that can potentiate S. aureus pathogenesis by facilitating bacterial survival within the neutrophil. These findings suggest avenues for novel immunomodulatory approaches to control S. aureus infections.
The Absence of Lymphoid CD8+ Dendritic Cell Maturation in L-Selectin−/− Respiratory Compartment Attenuates Antiviral Immunity
Authors: Pascual David W. , Wang Xinhai , Kochetkova Irina , Callis Gayle , Riccardi Carol
Published: April 2014

Intratracheal instillation of L-selectin-deficient (L-Sel−/−) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or β-galactosidase-specific CTLs with concomitant long-lived β-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c+CD8+ dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel−/− CTL activity, cell-sorted L-Sel−/−CD8+ T cells were cocultured with cell-sorted L-Sel+/+CD8+ or CD8− DCs or L-Sel−/−CD8− DCs. Only the CD8+ DCs restored CTL activity; L-Sel−/−CD8− DCs failed to support L-Sel+/+ CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8+ DCs were detected, the DC environment remained suppressive in L-Sel−/− mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel−/− adoptively transferred with OT-1 CD8+ T cells. To assess whether the L-Sel−/−CD8− DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel−/− LRLN to make matured DCs. When L-Sel−/− mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8+ DCs in L-Sel−/− mice. These studies show that impairment of L-Sel−/− DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8+ DCs to support CTL responses.
Granzymes Drive a Rapid Listeriolysin O-Induced T Cell Apoptosis
Authors: Carrero Javier A. , Vivanco-Cid Hector , Unanue Emil R.
Published: April 2014

The Listeria monocytogenes protein listeriolysin O (LLO) is a pore-forming protein essential for virulence. Although the major role for LLO is to allow L. monocytogenes entry into the cytosol, it also induces apoptosis of activated lymphocytes, an obligatory cellular response that modulates the infection. Induction of apoptosis by LLO proceeds through a fast, caspase-dependent pathway and a slow, caspase-independent pathway. Polyclonal T cell lines were generated from either normal mice or mice deficient in granzyme and perforin proteins, and then treated with apoptogenic doses of LLO. In this study we show that apoptosis of lymphocytes induced by LLO was characterized by activation of caspases as quickly as 30 min that was dependent on the expression of granzymes. In the absence of granzymes, all parameters of apoptosis such as caspase activation, phosphatidylserine exposure, mitochondrial depolarization, and DNA fragmentation were dramatically reduced in magnitude. Removal of perforin inhibited the apoptotic effect of LLO on cells by ∼50%. Neutralization of intracellular acidification using chloroquine inhibited the rapid apoptotic death. In agreement with these findings granzyme-deficient mice harbored lower bacterial titers and decrease splenic pathology compared with normal mice following L. monocytogenes infection. Thus, LLO exploits apoptotic enzymes of the adaptive immune response to eliminate immune cells and increase its virulence.
CD11c Expression Identifies a Population of Extrafollicular Antigen-Specific Splenic Plasmablasts Responsible for CD4 T-Independent Antibody Responses during Intracellular Bacterial Infection
Authors: Racine Rachael , Chatterjee Madhumouli , Winslow Gary M.
Published: April 2014

Although T-independent immunity is known to be generated against bacterial capsular and cell wall polysaccharides expressed by a number of bacterial pathogens, it has not been studied in depth during intracellular bacterial infections. Our previous study demonstrated that Ehrlichia muris, an obligate intracellular tick-borne pathogen, generates protective classical TI responses in CD4 T cell-deficient C57BL/6 mice. We found that E. muris T-independent immunity is accompanied by the expansion of a very large extrafollicular spleen population of CD11clow-expressing plasmablasts that exhibit characteristics of both B-1 and marginal zone B cells. The plasmablasts comprised up to 15% of the total spleen lymphocytes and ∼70% of total spleen IgMhighIgDlow cells during peak infection in both wild-type and MHC class II-deficient mice. The CD11clow cells exhibited low surface expression of B220, CD19, and CD1d, high expression of CD11b, CD43, but did not express CD5. Approximately 50% of the CD11clow cells also expressed CD138. In addition to CD11b and CD11c, the plasmablasts expressed the β1 (CD29) and α4 (CD49d) integrins, as well as the chemokine receptor CXCR4, molecules which may play roles in localizing the B cells extrafollicular region of the spleen. During peak infection, the CD11clow cells accounted for the majority of the IgM-producing splenic B cells and nearly all of the E. muris outer membrane protein-specific IgM-secreting cells. Thus, during this intracellular bacterial infection, CD11c expression identifies a population of Ag-specific spleen plasmablasts responsible for T-independent Ab production.
Complete Cure of Experimental Visceral Leishmaniasis with Amphotericin B in Stearylamine-Bearing Cationic Liposomes Involves Down-Regulation of IL-10 and Favorable T Cell Responses
Authors: Banerjee Antara , De Manjarika , Ali Nahid
Published: April 2014

Visceral leishmaniasis caused by Leishmania donovani is a life-threatening disease involving uncontrolled parasitization of liver, spleen, and bone marrow. Most available drugs are toxic. Moreover, relapse after seemingly successful therapy remains a chronic problem. In this study, we evaluated a new therapeutic approach based on combination of a low dose of amphotericin B (AmB) in association with suboptimum dose of stearylamine (SA)-bearing cationic liposomes, itself having leishmanicidal activity. We demonstrate that a single-shot therapy with this formulation caused clearance of parasites from liver and spleen below the level of detection in the selected piece of the organs of BALB/c mice. The combination was superior to free AmB and AmBisome for therapy, as well as for prevention of relapse and reinfection. Besides having better killing activity, AmB in SA liposomes, in contrast to AmBisome, maintained the immunomodulatory effect of free AmB on CD4+ and CD8+ T cells for IFN-γ production, at the same time reducing the toxic effects of the drug, reflected through decline in TNF-α. In addition, IL-10 was down-regulated to almost negligible levels, most efficiently through therapy with SA-bearing cationic liposomes-AmB. This IL-10-deficient environment of IFN-γ-secreting T cells probably up-regulated the enhanced IL-12 and NO production observed in splenic culture supernatants of these mice, correlating with prolonged disease suppression better than free AmB and AmBisome. The ability of the formulation to elicit protective immunity was reconfirmed in a prophylactic model. Our results emphasize the requirement of effective immune stimulation, additionally, by antileishmanials for persistent disease protection, demonstrated by this liposomal AmB formulation.
Apolipoprotein E-Mediated Immune Regulation in Sepsis
Authors: Kattan Omar M. , Kasravi F. Behzad , Elford Erica L. , Schell Michael T. , Harris Hobart W.
Published: April 2014

Lipids and lipoproteins have emerged as key constituents of the immune response to microbial infection. We, therefore, sought to understand the complex interaction between lipoprotein metabolism and sepsis. Apolipoprotein E (apoE), a component of plasma lipoproteins, has been suggested to bind and traffic Ags for NKT cell activation. However, apoE’s role in sepsis has not been demonstrated. In this study, we examined the effect of exogenous apoE in a rat model of septic peritonitis, induced by cecal ligation and puncture. We demonstrate that 48 h after serial injections of apoE, septic mortality increased in a dose-dependent manner. While sepsis resulted in increased splenic and decreased hepatic and circulating NKT cell populations, serial injections of apoE for 24 h after cecal ligation and puncture increased the frequency, cell number, and BrdU uptake in splenic and hepatic NKT cell populations, while concomitantly depleting these populations in the circulation. These changes were correlated with elevated alanine amino transferase levels, an indicator of liver injury. Interestingly, while sepsis increased hepatic T cell apoptosis and necrosis, apoE reversed these changes. apoE also promoted increases in predominantly Th1 cytokine levels in sera and a decrease in IL-4, the main NKT cell-derived Th2 cytokine. Consequently, apoE treatment is associated with increased sepsis-induced mortality, and increased NKT cell frequency and proliferation in the liver and spleen, with concomitant decreases in these NKT cell parameters in the peripheral circulation. apoE treatment also promoted a Th1 cytokine response, increased the degree of liver injury, and decreased apoptosis in hepatic lymphocytes.
Identification of Formyl Peptides from Listeria monocytogenes and Staphylococcus aureus as Potent Chemoattractants for Mouse Neutrophils
Authors: Southgate Erica L. , He Rong L. , Gao Ji-Liang , Murphy Philip M. , Nanamori Masakatsu , Ye Richard D.
Published: April 2014

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1–10 nM and superoxide production at 10–100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are ∼100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.
Varying Importance of Soluble and Membrane CD14 in Endothelial Detection of Lipopolysaccharide
Authors: Lloyd-Jones Katie L. , Kelly Margaret M. , Kubes Paul
Published: April 2014

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-β) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.
Fractalkine and CX3CR1 Mediate Leukocyte Capture by Endothelium in Response to Shiga Toxin
Authors: Zanchi Cristina , Zoja Carla , Morigi Marina , Valsecchi Federica , Liu Xue Yan , Rottoli Daniela , Locatelli Monica , Buelli Simona , Pezzotta Anna , Mapelli Paola , Geelen Joyce , Remuzzi Giuseppe , Hawiger Jacek
Published: April 2014

Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-κB and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IκBα inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.
Monocyte Chemoattractant Protein-1 (CCL-2) Integrates Mechanical and Endocrine Signals That Mediate Term and Preterm Labor
Authors: Shynlova Oksana , Tsui Prudence , Dorogin Anna , Lye Stephen J.
Published: April 2014

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 μM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.
Endoglycan, a Member of the CD34 Family of Sialomucins, Is a Ligand for the Vascular Selectins
Authors: Kerr Sheena C. , Fieger Claudia B. , Snapp Karen R. , Rosen Steven D.
Published: April 2014

The interactions of the selectin family of adhesion molecules with their ligands are essential for the initial rolling stage of leukocyte trafficking. Under inflammatory conditions, the vascular selectins, E- and P-selectin, are expressed on activated vessels and interact with carbohydrate-based ligands on the leukocyte surface. While several ligands have been characterized on human T cells, monocytes and neutrophils, there is limited information concerning ligands on B cells. Endoglycan (EG) together with CD34 and podocalyxin comprise the CD34 family of sialomucins. We found that EG, previously implicated as an L-selectin ligand on endothelial cells, was present on human B cells, T cells and peripheral blood monocytes. Upon activation of B cells, EG increased with a concurrent decrease in PSGL-1. Expression of EG on T cells remained constant under the same conditions. We further found that native EG from several sources (a B cell line, a monocyte line and human tonsils) was reactive with HECA-452, a mAb that recognizes sialyl Lewis X and related structures. Moreover, immunopurified EG from these sources was able to bind to P-selectin and where tested E-selectin. This interaction was divalent cation-dependent and required sialylation of EG. Finally, an EG construct supported slow rolling of E- and P-selectin bearing cells in a sialic acid and fucose dependent manner, and the introduction of intact EG into a B cell line facilitated rolling interactions on a P-selectin substratum. These in vitro findings indicate that EG can function as a ligand for the vascular selectins.
Novel Immunomodulatory Properties of Berbamine through Selective Down-Regulation of STAT4 and Action of IFN-γ in Experimental Autoimmune Encephalomyelitis
Authors: Ren Yiping , Lu Limin , Guo Taylor B. , Qiu Ju , Yang Yiqing , Liu Ailian , Zhang Jingwu Z.
Published: April 2014

Berbamine (BM) is an herbal compound derived from Berberis vulgaris L commonly used in traditional Chinese medicine. In this study, we show that BM has potent anti-inflammatory properties through novel regulatory mechanisms, leading to reduced encephalitogenic T cell responses and amelioration of experimental autoimmune encephalomyelitis (EAE). The treatment effect of BM was attributable to its selective inhibitory effect on the production and action of IFN-γ in CD4+ T cells, which was mediated through altered STAT4 expression in T cells. BM was found to up-regulate SLIM, a ubiquitin E3 ligase for STAT4, and promote STAT4 degradation, resulting in markedly decreased IFN-γ production in CD4+ T cells in EAE mice. Regulation of IFN-γ by BM had profound anti-inflammatory actions through its effect on both CD4+ T cells and APCs. BM-treated APCs exhibited reduced stimulatory function as a result of altered expression of PD-L1, CD80, and CD86 in treated mice. The treatment effect of BM in EAE was directly related to its action on IFN-γ, and was abolished in IFN-γ knockout mice. The study also confirmed that BM was able to inhibit NFAT translocation through effecting calcium mobilization in lymphocytes. However, this effect was not directly responsible for the treatment efficacy of BM in EAE. The study has important implications in our approaches to evaluating the utility of natural compounds in drug discovery and to probing the role of cytokine network in the development of autoimmune conditions.
IL-23 Induces Receptor Activator of NF-κB Ligand Expression on CD4+ T Cells and Promotes Osteoclastogenesis in an Autoimmune Arthritis Model
Authors: Ju Ji Hyeon , Cho Mi-La , Moon Young-Mee , Oh Hye-Joa , Park Jin-Sil , Jhun Joo-Youn , Min So-Youn , Cho Young-Gyu , Park Kyung-Su , Yoon Chong-Hyeon , Min Jun-Ki , Park Sung-Hwan , Sung Young-Chul , Kim Ho-Youn
Published: April 2014

IL-23, a clinically novel cytokine, targets CD4+ T cells. Recent IL-1Ra−/− mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4+ T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-κB ligand expression by CD4+ T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-κB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra−/− mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4+ T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.
Cigarette Smoke-Induced Oxidative Stress Suppresses Generation of Dendritic Cell IL-12 and IL-23 through ERK-Dependent Pathways
Authors: Kroening Paula R. , Barnes Terrance W. , Pease Larry , Limper Andrew , Kita Hirohito , Vassallo Robert
Published: April 2014

IL-12p70, a heterodimer composed of p35 and p40 subunits, is a key polarizing cytokine produced by maturing dendritic cells (DCs). We report that cigarette smoke extract (CSE), an extract of soluble cigarette smoke components, suppresses both p35 and p40 production by LPS or CD40L-matured DCs. Suppression of IL-12p70 production from maturing DCs was not observed in the presence of nicotine concentrations achievable in CSE or in the circulation of smokers. The suppressed IL-12p70 protein production by CSE-conditioned DCs was restored by pretreatment of DCs or CSE with the antioxidants N-acetylcysteine and catalase. Inhibition of DC IL-12p70 by CSE required activation of ERK-dependent pathways, since inhibition of ERK abrogated the suppressive effect of CSE on IL-12 secretion. Oxidative stress and sustained ERK phosphorylation by CSE enhanced nuclear levels of the p40 transcriptional repressor c-fos in both immature and maturing DCs. Suppression of the p40 subunit by CSE also resulted in diminished production of IL-23 protein by maturing DCs. Using a murine model of chronic cigarette smoke exposure, we observed that systemic and lung DCs from mice “smokers” produced significantly less IL-12p70 and p40 protein upon maturation. This inhibitory effect was selective, since production of TNF-α during DC maturation was enhanced in the smokers. These data imply that oxidative stress generated by cigarette smoke exposure suppresses the generation of key cytokines by maturing DCs through the activation of ERK-dependent pathways. Some of the cigarette smoke-induced inhibitory effects on DC function may be mitigated by antioxidants.