bluuebunny/crossref_metadata_2025 huggingface dataset:
Central Role of MyD88-Dependent Dendritic Cell Maturation and Proinflammatory Cytokine Production to Control Brucella abortus Infection
Authors: Macedo Gilson Costa , Magnani Diogo Matos , Carvalho Natalia Barbosa , Bruna-Romero Oscar , Gazzinelli Ricardo T. , Oliveira Sergio Costa
Published: April 2014

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-α production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-α administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-α, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-α production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-γ.
Regulation of IL-18 inHelicobacter pyloriInfection
Authors: Yamauchi Kazuyoshi , Choi Il-Ju , Lu Hong , Ogiwara Hiroaki , Graham David Y. , Yamaoka Yoshio
Published: April 2014

The gastric mucosal immune response is thought to be comprised predominantly of the Th1 type; however, there are limited data regarding the role of IL-18 in Helicobacter pylori-induced inflammation. We investigated IL-18 levels in gastric mucosal biopsy specimens as well as in isolated gastric epithelial cells and lamina propria mononuclear cells. We also investigated IL-18 levels in gastric epithelial cells and the monocyte cell line THP-1 cocultured with H. pylori. In both systems, IL-18 levels were markedly enhanced in H. pylori-infected epithelial cells and monocytes. IL-18 levels in H. pylori-infected gastric mucosa were well correlated with the severity of gastric inflammation, confirming that H. pylori-induced IL-18 plays an important role in gastric injury. Virulence factors of H. pylori; the cag pathogenicity island and OipA affected IL-18 induction in different manners. Up-regulation of IL-18 mRNA/protein in epithelial cells was dependent on both virulence factors. Interestingly, up-regulation of IL-18 mRNA in monocytes was independent of both factors, whereas IL-18 protein was OipA dependent – cag pathogenicity island independent, indicating that OipA regulates IL-18 induction in monocytes at the posttranscriptional level. IL-18 levels in the gastric biopsy specimens showed similar patterns to those in lamina propria mononuclear cells with respect to virulence factors, suggesting that submucosal monocytes/macrophages are the main source of IL-18 induced by H. pylori infection. H. pylori appeared to regulate the ERK/JNK→AP-1 pathway in both cell types. In addition, OipA and its related p38 pathway may be closely involved in IL-18 induction in H. pylori-infected gastric mucosa and may contribute to gastric injury.
Apoptotic Cell-Derived Sphingosine-1-Phosphate Promotes HuR-Dependent Cyclooxygenase-2 mRNA Stabilization and Protein Expression
Authors: Johann Axel M. , Weigert Andreas , Eberhardt Wolfgang , Kuhn Anne-Marie , Barra Vera , von Knethen Andreas , Pfeilschifter Josef M. , Brüne Bernhard
Published: April 2014

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3′-untranslated region (UTR), shown by COX-2–3′-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.
Expression and Regulation of CCR1 by Airway Smooth Muscle Cells in Asthma
Authors: Joubert Philippe , Lajoie-Kadoch Stéphane , Welman Mélanie , Dragon Stephane , Létuvée Séverine , Tolloczko Barbara , Halayko Andrew J. , Gounni Abdelilah Soussi , Maghni Karim , Hamid Qutayba
Published: April 2014

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-α and, to a lesser extent, IFN-γ resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.
Cutting Edge: T-bet and IL-27R Are Critical for In Vivo IFN-γ Production by CD8 T Cells during Infection
Authors: Mayer Katrin D. , Mohrs Katja , Reiley William , Wittmer Susan , Kohlmeier Jacob E. , Pearl John E. , Cooper Andrea M. , Johnson Lawrence L. , Woodland David L. , Mohrs Markus
Published: April 2014

CD8+ T cells are a major source of IFN-γ, a key effector cytokine in immune responses against many viruses and protozoa. Although the transcription factor T-bet is required for IFN-γ expression in CD4+ T cells, it is reportedly dispensable in CD8+ T cells, where the transcription factor Eomesodermin is thought to be sufficient. The diverse functions of IFN-γ are mediated through the IFN-γR and STAT1. In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop. However, STAT1 can also be activated by other cytokines, including IL-27. In the present study we show that, in contrast to in vitro conditions and the prevailing paradigm, T-bet is critical for the in vivo IFN-γ production by CD8+ T cells upon infection of mice with diverse pathogens. Whereas IFN-γR signals are dispensable for the T-bet-dependent IFN-γ production, direct IL-27Rα signals are critical.
Transgenic CD4 T Cells (DO11.10) Are Destroyed in MHC-Compatible Hosts by NK Cells and CD8 T Cells
Authors: Duffy Darragh , Sparshott Sheila M. , Yang Chun-ping , Bell Eric B.
Published: April 2014

During an immune response a small number of rare Ag-specific clones proliferate extensively and decline, leaving a residual population for long-term memory. TCR transgenic (tg) CD4 T cells have been used widely to study the primary and memory response in vivo. We show here that naive TCR tg CD4 T cells from the DO11.10 strain transferred into wild type (wt) BALB/c recipients and not stimulated declined rapidly at the same rate as those primed in vivo by Ag. In the same recipients wt CD4 T cells survived. There was no evidence of an inherent defect in the tg T cells, which survived well when returned to DO11.10 recipients. Surprisingly, wt CD4 T cells declined rapidly in the same DO11.10 hosts. By depleting wt recipients of NK cells or CD8+ cells, the speed of reduction was slowed by half; rapid destruction was prevented completely by combing the two treatments. In contrast, preimmunization accelerated the loss of tg T cells. The results suggested that tg CD4 T cells were actively rejected by both NK and CD8 T cell responses. We consider whether, despite extensive backcrossing, tg T cells may retain genetic material (minor histocompatibility Ags) flanking the construct that compromises their survival in wt recipients.
Naive Precursors of Human Regulatory T Cells Require FoxP3 for Suppression and Are Susceptible to HIV Infection
Authors: Antons Amanda K. , Wang Rui , Oswald-Richter Kyra , Tseng Michelle , Arendt Christopher W. , Kalams Spyros A. , Unutmaz Derya
Published: April 2014

CD4+CD25+ human regulatory T cells (Treg cells), which express the transcription factor FoxP3, suppress T cell activation. In this study, we sought to define cellular and molecular mechanisms of human Treg cell differentiation. A subset of human naive CD4+ T cells that are CD25+ express high levels of FoxP3. We show that upon activation through the TCR, these FoxP3-expressing naive T cells (termed TNreg cells) greatly expand in vitro. Expanded TNreg cells acquire a full Treg phenotype with potent suppressive activity and display low IL-2 production upon TCR stimulation. TNreg cells in which FoxP3 expression was reduced through RNA interference lost their suppressive activity, but retained their low IL-2 secretion in response to TCR stimulation. Furthermore, in support of the notion that TNreg cells represent a separate lineage of naive cells, we found that they were more susceptible to HIV infection as compared with naive CD4+ T cells. Based on these findings, we propose that TNreg cells are precursors for human Treg cells and that these cells require a high level of FoxP3 expression to maintain their suppressive function. Accordingly, modulation of TNreg cell numbers during infections such as HIV may disrupt human Treg cell development, and contribute to chronic immune activation.
Live Cell Imaging Reveals that the Inhibitory FcγRIIB Destabilizes B Cell Receptor Membrane-Lipid Interactions and Blocks Immune Synapse Formation
Authors: Sohn Hae Won , Pierce Susan K. , Tzeng Shiang-Jong
Published: April 2014

The FcγRIIB is a potent regulator of BCR signaling and as such plays a decisive role in controlling autoimmunity. The use of advanced imaging technologies has provided evidence that the earliest events in Ag-induced BCR signaling include the clustering of the BCR, the selective and transient association of the clustered BCR with raft lipids, and the concentration of BCR clusters in an immune synapse. That lipid rafts play a role in FcγRIIB’s regulation of BCR signaling was suggested by recent studies showing that a lupus-associated loss of function mutation resulted in the receptor’s exclusion from lipid rafts and the failure to regulate BCR signaling. In this study, we provide evidence from both biochemical analyses and fluorescence resonance energy transfer in conjunction with both confocal and total internal reflection microscopy in living cells that the FcγRIIB, when coligated with the BCR, associates with lipid rafts and functions both to destabilize the association of the BCR with raft lipids and to block the subsequent formation of the B cell’s immune synapse. These results define new early targets of FcγRIIB inhibitory activity in the Ag-induced B cell activation pathway.
In Vitro Expansion Improves In Vivo Regulation by CD4+CD25+ Regulatory T Cells
Authors: Chai Jian-Guo , Coe David , Chen Daxin , Simpson Elizabeth , Dyson Julian , Scott Diane
Published: April 2014

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFβ while down-regulating IFN-γ, GM-CSF, IL5, and TNF-α production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFβ to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.
Intranasal Vaccination of Recombinant Adeno-Associated Virus Encoding Receptor-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Spike Protein Induces Strong Mucosal Immune Responses and Provides Long-Term Protection against SARS-CoV Infection
Authors: Du Lanying , Zhao Guangyu , Lin Yongping , Sui Hongyan , Chan Chris , Ma Selene , He Yuxian , Jiang Shibo , Wu Changyou , Yuen Kwok-Yung , Jin Dong-Yan , Zhou Yusen , Zheng Bo-Jian
Published: April 2014

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-γ-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.
Protein Interactions between CD2 and Lck Are Required for the Lipid Raft Distribution of CD2
Authors: Nunes Raquel J. , Castro Mónica A. A. , Gonçalves Carine M. , Bamberger Martina , Pereira Carlos F. , Bismuth Georges , Carmo Alexandre M.
Published: April 2014

In T lymphocytes, lipid rafts are preferred sites for signal transduction initiation and amplification. Many cell membrane receptors, such as the TCR, coreceptors, and accessory molecules associate within these microdomains upon cell activation. However, it is still unclear in most cases whether these receptors interact with rafts through lipid-based amino acid modifications or whether raft insertion is driven by protein-protein interactions. In murine T cells, a significant fraction of CD2 associates with membrane lipid rafts. We have addressed the mechanisms that control the localization of rat CD2 at the plasma membrane, and its redistribution within lipid rafts induced upon activation. Following incubation of rat CD2-expressing cells with radioactive-labeled palmitic acid, or using CD2 mutants with Cys226 and Cys228 replaced by alanine residues, we found no evidence that rat CD2 was subjected to lipid modifications that could favor the translocation to lipid rafts, discarding palmitoylation as the principal mechanism for raft addressing. In contrast, using Jurkat cells expressing different CD2 and Lck mutants, we show that the association of CD2 with the rafts fully correlates with CD2 capacity to bind to Lck. As CD2 physically interacts with both Lck and Fyn, preferentially inside lipid rafts, and reflecting the increase of CD2 in lipid rafts following activation, CD2 can mediate the interaction between the two kinases and the consequent boost in kinase activity in lipid rafts.
Expression of Aire and the Early Wave of Apoptosis in Spermatogenesis
Authors: Schaller Claudia E. , Wang Clifford L. , Beck-Engeser Gabriele , Goss Lindsie , Scott Hamish S. , Anderson Mark S. , Wabl Matthias
Published: April 2014

Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted. In this study, we show that Aire mRNA is also expressed in the testis—another tissue with promiscuous gene expression. Aire protein, however, is expressed only sporadically in spermatogonia and spermatocytes. Transcription of genes that are under Aire control in the thymus is unaffected by Aire in the testis. However, in mice with a disrupted Aire gene, the scheduled apoptotic wave of germ cells, which is necessary for normal mature spermatogenesis, is reduced, and sporadic apoptosis in adults is increased. Because Rag-1 deficiency does not abolish the effect, the adaptive immune system is not involved. We suggest that there is a link between the scheduled and sporadic apoptotic processes and propose that scheduled apoptosis provides a counterselection mechanism that keeps the germline stable.
Autoantigen-Specific IL-10-Transduced T Cells Suppress Chronic Arthritis by Promoting the Endogenous Regulatory IL-10 Response
Authors: Guichelaar Teun , ten Brink Corlinda B. , van Kooten Peter J. , Berlo Suzanne E. , Broeren Chris P. , van Eden Willem , Broere Femke
Published: April 2014

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4+ T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4+ T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4+ T cells transduced with an active IL-10 gene (TIL-10) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific TIL-10 cells ameliorated arthritis, whereas TIL-10 cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific TIL-10 cells suppressed autoreactive proinflammatory T and B cells, as TIL-10 cells caused a reduced expression of IL-2, TNF-α, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific TIL-10 cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that TIL-10 cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.
Route of Uptake of Palmitoylated Encephalitogenic Peptides of Myelin Proteolipid Protein by Antigen-Presenting Cells: Importance of the Type of Bond between Lipid Chain and Peptide and Relevance to Autoimmunity
Authors: Pfender Nadège A. , Grosch Sylvie , Roussel Guy , Koch Marc , Trifilieff Elisabeth , Greer Judith M.
Published: April 2014

Previously, we have shown that thiopalmitoylation of peptides of myelin proteolipid protein, as occurs naturally in vivo, increases their ability to induce experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and skews the autoimmune response toward a CD4+-mediated response. In contrast, the same peptide, when synthesized with a stable amide bond between peptide and lipid, inhibits experimental autoimmune encephalomyelitis and skews the response toward a CD8+ response. The aim of the current study was to determine the mechanisms responsible for these observations. We show that proteolipid protein lipopeptides, when synthesized with a thioester bond between the lipid and the peptide, are taken up into APCs via an actin-independent endocytic route, the thioester bond is cleaved in the endosome, and the peptide is subsequently displayed on the surface of the APC in the context of MHC class II. The same peptide, when synthesized with the lipid attached via a stable amide bond, rapidly enters into the cytoplasm of the APC and forms micelles; however, the bond between peptide and lipid is not cleaved, and the micelles travel via the endoplasmic reticulum to complex with MHC class I. These findings have implications for vaccine development and for the development of MHC class II-restricted autoimmune diseases, as many human autoantigens thus far identified are thioacylated.
In Trans T Cell Tolerance Diminishes Autoantibody Responses and Exacerbates Experimental Allergic Encephalomyelitis
Authors: Bell J. Jeremiah , Divekar Rohit D. , Ellis Jason S. , Cascio Jason A. , Haymaker Cara L. , Jain Renu , Tartar Danielle M. , Hoeman Christine M. , Hardaway John C. , Zaghouani Habib
Published: April 2014

A number of Ag-specific approaches have been developed that ameliorate experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis. Translation to humans, however, remains a consideration, justifying the search for more insight into the mechanism underlying restoration of self-tolerance. Ig-proteolipid protein (PLP) 1 and Ig-myelin oligodendrocyte glycoprotein (MOG) are Ig chimeras carrying the encephalitogenic PLP 139–151 and MOG 35–55 amino acid sequence, respectively. Ig-PLP1 ameliorates EAE in SJL/J (H-2s) mice while Ig-MOG modulates the disease in C57BL/6 (H-2b) animals. In this study, we asked whether the chimeras would suppress EAE in F1 mice expressing both parental MHC alleles and representing a polymorphism with more relevance to human circumstances. The results show that Ig-MOG modulates both PLP1 and MOG peptide-induced EAE in the F1 mice, whereas Ig-PLP1 counters PLP1 EAE but exacerbates MOG-induced disease. This in trans aggravation of MOG EAE by Ig-PLP1 operates through induction of PLP1-specific T cells producing IL-5 that sustained inhibition of MOG-specific Abs leading to exacerbation of EAE. Thus, in trans T cell tolerance, which should be operative in polymorphic systems, can aggravate rather than ameliorate autoimmunity. This phenomenon possibly takes place through interference with protective humoral immunity.
T Regulatory Cells Contribute to the Attenuated Primary CD8+ and CD4+ T Cell Responses to Herpes Simplex Virus Type 2 in Neonatal Mice
Authors: Fernandez Marian A. , Puttur Franz K. , Wang Yuan M. , Howden Wade , Alexander Stephen I. , Jones Cheryl A.
Published: April 2014

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4+Foxp3+ Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8+ T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8+ and CD4+ T cell IFN-γ responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-β in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.
An MHC Class II Restriction Bias in CD4 T Cell Responses toward I-A Is Altered to I-E in DM-Deficient Mice
Authors: Menges Paula R. , Jenks Scott A. , Bikoff Elizabeth K. , Friedmann David R. , Knowlden Zackery A. G. , Sant Andrea J.
Published: April 2014

The MHC-encoded cofactor DM catalyzes endosomal loading of peptides onto MHC class II molecules. Despite evidence from in vitro experiments that DM acts to selectively edit the repertoire of class II:peptide complexes, the consequence of DM expression in vivo, or a predictive pattern of DM activity in the specificity of CD4 T cell responses has remained unresolved. Therefore, to characterize DM function in vivo we used wild-type (WT) or DM-deficient (DM−/−) mice of the H-2d MHC haplotype and tested the hypothesis that DM promotes narrowing of the repertoire of class II:peptide complexes displayed by APC, leading to a correspondingly selective CD4 T cell response. Surprisingly, our results indicated that DM−/− mice do not exhibit a broadened CD4 T cell response relative to WT mice, but rather shift their immunodominance pattern to new peptides, a pattern associated with a change in class II isotype-restriction. Specifically, we found that CD4 T cell responses in WT mice were primarily restricted to the I-A class II molecule, whereas DM−/− mice recognize peptides in the context of I-E. The observed shift in isotype-restriction appeared to be due in part to a modification in the peripheral CD4 T cell repertoire available for peptide recognition.
Two Novel NKG2D Ligands of the Mouse H60 Family with Differential Expression Patterns and Binding Affinities to NKG2D
Authors: Takada Akio , Yoshida Shigeru , Kajikawa Mizuho , Miyatake Yukiko , Tomaru Utano , Sakai Masaharu , Chiba Hitoshi , Maenaka Katsumi , Kohda Daisuke , Fugo Kazunori , Kasahara Masanori
Published: April 2014

H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell–cell recognition receptors (Kd = 310 nM for H60b and Kd = 8.7 μM for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby establishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands.
Identification of Cellular Pathways of “Type 1,” Th17 T Cells, and TNF- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells in Autoimmune Inflammation through Pharmacogenomic Study of Cyclosporine A in Psoriasis
Authors: Haider Asifa S. , Lowes Michelle A. , Suárez-Fariñas Mayte , Zaba Lisa C. , Cardinale Irma , Khatcherian Artemis , Novitskaya Inna , Wittkowski Knut M. , Krueger James G.
Published: April 2014

Therapeutic modulation of psoriasis with targeted immunosuppressive agents defines inflammatory genes associated with disease activity and may be extrapolated to a wide range of autoimmune diseases. Cyclosporine A (CSA) is considered a “gold standard” therapy for moderate-to-severe psoriasis. We conducted a clinical trial with CSA and analyzed the treatment outcome in blood and skin of 11 responding patients. In the skin, as expected, CSA modulated genes from activated T cells and the “type 1” pathway (p40, IFN-γ, and STAT-1-regulated genes). However, CSA also modulated genes from the newly described Th17 pathway (IL-17, IL-22, and downstream genes S100A12, DEFB-2, IL-1β, SEPRINB3, LCN2, and CCL20). CSA also affected dendritic cells, reducing TNF and inducible NO synthase (products of inflammatory TNF- and inducible NO synthase-producing dendritic cells), CD83, and IL-23p19. We detected 220 early response genes (day 14 posttreatment) that were down-regulated by CSA. We classified >95% into proinflammatory or skin resident cells. More myeloid-derived than activated T cell genes were modulated by CSA (54 myeloid genes compared with 11 lymphocyte genes), supporting the hypothesis that myeloid derived genes contribute to pathogenic inflammation in psoriasis. In circulating mononuclear leukocytes, in stark contrast, no inflammatory gene activity was detected. Thus, we have constructed a genomic signature of successful treatment of psoriasis which may serve as a reference to guide development of other new therapies. In addition, these data also identify new gene targets for therapeutic modulation and may be applied to wide range of autoimmune diseases.
Identification of a Molecular Signature in Human Type 1 Diabetes Mellitus Using Serum and Functional Genomics
Authors: Wang Xujing , Jia Shuang , Geoffrey Rhonda , Alemzadeh Ramin , Ghosh Soumitra , Hessner Martin J.
Published: April 2014

Understanding active proinflammatory mechanisms at and before type 1 diabetes mellitus (T1DM) onset is hindered in humans, given that the relevant tissues are inaccessible and pancreatic immune responses are difficult to measure in the periphery by traditional approaches. Therefore, we investigated the use of a sensitive and comprehensive genomics strategy to investigate the presence of proinflammatory factors in serum. The sera of recent onset diabetes patients (n = 15, 12 possessing and 3 lacking islet cell autoantibodies), long-standing diabetes patients (n = 12), “at risk” siblings of diabetes patients (n = 9), and healthy controls (n = 12) were used to induce gene expression in unrelated, healthy PBMC. After culture, gene expression was measured with microarrays and normalized expression data were subjected to hierarchical clustering and multidimensional scaling. All recent onset sera induced an expression signature (192 UniGenes; fold change: >1.5, p < 0.01; false discovery rate: <0.01) that included IL-1 cytokine family members and chemokines involved in monocyte/macrophage and neutrophil chemotaxis, as well as numerous receptors and signaling molecules. This molecular signature was not induced with the sera of healthy controls or long standing diabetes patients, where longitudinal analysis of “at risk” siblings (n = 3) before and after onset support the hypothesis that the signature emerges years before onset. This study supports prior investigations of serum that reflect disease processes associated with progression to T1DM. Identification of unique inflammatory mediators may improve disease prediction beyond current islet autoantibodies. Furthermore, proinflammatory serum markers may be used as inclusion criteria or endpoint measures in clinical trials aimed at preventing T1DM.
Successful Treatment of Animal Models of Rheumatoid Arthritis with Small-Molecule Cyclin-Dependent Kinase Inhibitors
Authors: Sekine Chiyoko , Sugihara Takahiko , Miyake Sachiko , Hirai Hiroshi , Yoshida Mitsuaki , Miyasaka Nobuyuki , Kohsaka Hitoshi
Published: April 2014

Intraarticular gene transfer of cyclin-dependent kinase (CDK) inhibitors to suppress synovial cell cycling has shown efficacy in treating animal models of rheumatoid arthritis. Endogenous CDK inhibitors also modulate immune function via a CDK-independent pathway. Accordingly, systemic administration of small molecules that inhibit CDK may or may not ameliorate arthritis. To address this issue, alvocidib (flavopiridol), known to be tolerated clinically for treating cancers, and a newly synthesized CDK4/6-selective inhibitor were tested for antiarthritic effects. In vitro, they inhibited proliferation of human and mouse synovial fibroblasts without inducing apoptosis. In vivo, treatment of collagen-induced arthritis mice with alvocidib suppressed synovial hyperplasia and joint destruction, whereas serum concentrations of anti-collagen type II (CII) Abs and proliferative responses to CII were maintained. Treatment was effective even when therapeutically administered. Treated mice developed arthritis after termination of treatment. Thus, immune responses to CII were unimpaired. The same treatment ameliorated arthritis induced by K/BxN serum transfer to lymphocyte-deficient mice. Similarly, the CDK4/6-selective inhibitor suppressed collagen-induced arthritis. Both small-molecule CDK inhibitors were effective in treating animal models of rheumatoid arthritis not by suppressing lymphocyte function. Thus, the two small-molecule CDK inhibitors ameliorated arthritis models in a distinctive way, compared with other immunosuppressive drugs.
Synoviocyte Stimulation by the LFA-1–Intercellular Adhesion Molecule-2–Ezrin–Akt Pathway in Rheumatoid Arthritis
Authors: Singh Karnail , Colmegna Inés , He Xiaowen , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

In rheumatoid arthritis (RA), the synovium is infiltrated by mononuclear cells that influence the proliferation and activation of fibroblast-like synoviocytes (FLS) through soluble mediators as well as cell-to-cell contact. To identify receptor-ligand pairs involved in this cross-talk, we cocultured T cells with FLS lines isolated from synovial tissues from RA patients. Coculture with T cells induced phosphorylation of Akt (Ser473) and its downstream mediators, GSK-3α/GSK-β, FoxO1/3a, and mouse double minute-2, and enhanced FLS proliferation. T cell-mediated phospho-Akt up-regulation was unique for FLS as no such effect was observed upon interaction of T cells with dendritic cells and B cells. Akt activation was induced by all functional T cell subsets independent of MHC/Ag recognition and was also found with other leukocyte populations, suggesting the involvement of a common leukocyte cell surface molecule. Akt phosphorylation, enhanced in vitro FLS proliferation, and enhanced FLS IL-6 production was inhibited by blocking Abs to CD11a and ICAM-2 whereas Abs to ICAM-1 had a lesser effect. Selective involvement of the LFA-1–ICAM-2 pathway was confirmed by the finding of increased ezrin phosphorylation at Tyr353 that is known to be downstream of ICAM-2 and supports cell survival through Akt activation. CD28− T cells, which are overrepresented in RA patients, have high CD11a cell surface expression and induce Akt phosphorylation in FLS more potently than their CD28+ counterparts. These findings identify ICAM-2 as a potential therapeutic target to inhibit FLS activation in RA, allowing for a more selective intervention than broad LFA-1 inhibition.
Kit Regulates Maintenance of Quiescent Hematopoietic Stem Cells
Authors: Thorén Lina A. , Liuba Karina , Bryder David , Nygren Jens M. , Jensen Christina T. , Qian Hong , Antonchuk Jennifer , Jacobsen Sten-Eirik W.
Published: April 2014

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (KitW41/W41) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult KitW41/W41 mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin−Sca-1+Kithigh (LSK)CD34−Flt3− long-term HSCs by 12 wk of age, whereas LSKCD34+Flt3− short-term HSCs and LSKCD34+Flt3+ multipotent progenitors were less affected. Whereas homing and initial reconstitution of KitW41/W41 bone marrow cells in myeloablated recipients were close to normal, self-renewing KitW41/W41 HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation KitW41/W41 HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.
Selective Regulation of CD8 Effector T Cell Migration by the p110γ Isoform of Phosphatidylinositol 3-Kinase
Authors: Martin Amanda L. , Schwartz Matthew D. , Jameson Stephen C. , Shimizu Yoji
Published: April 2014

Chemokine-mediated T cell migration is essential to an optimal immune response. The p110γ isoform of PI3K is activated by G protein-coupled receptors and regulates neutrophil and macrophage chemotaxis. We used p110γ-deficient mice to examine the role of p110γ in CD8 T cell migration and activation in response to viral challenge. Naive CD8 T cell migration in response to CCL21 in vitro and trafficking into secondary lymphoid organs in vivo was unaffected by the loss of p110γ. Furthermore, loss of p110γ did not affect CD8 T cell proliferation and effector cell differentiation in vitro in response to anti-CD3 stimulation or in vivo in response to vaccinia virus (VV) challenge. However, there was reduced migration of p110γ knockout (p110γ−/−) CD8 effector T cells into the peritoneum following i.p. challenge with VV. The role of p110γ in CD8 effector T cell migration was intrinsic to T cells, as p110γ−/− CD8 effector T cells exhibited impaired migration into the inflamed peritoneum following secondary transfer into wild-type recipients. In addition, p110γ−/− CD8 effector T cells exhibited impaired migration in vitro in response to inflammatory chemoattractants. Although wild-type mice efficiently cleared VV at high viral doses, infection of p110γ knockout mice resulted in visible illness and death less than a week after infection. Thus, p110γ is dispensable for constitutive migration of naive CD8 T cells and subsequent activation and differentiation into effector CD8 T cells, but plays a central role in the migration of effector CD8 T cells into inflammatory sites.
STAT1 Signaling in CD8 T Cells Is Required for Their Clonal Expansion and Memory Formation Following Viral Infection In Vivo
Authors: Quigley Michael , Huang Xiaopei , Yang Yiping
Published: April 2014

Recent advances have shown that direct type I IFN signaling on T cells is required for their efficient expansion in response to viral infections in vivo. It is not clear which intracellular signaling molecule is responsible for this effect. Although STAT1 has been shown to mediate many of the type I IFN-dependent biological effects, its role in T cells remains uncertain in vivo. In this study, we demonstrated that STAT1 signaling in CD8 T cells was required for their efficient expansion by promoting the survival of activated CD8 T cells upon vaccinia viral infection in vivo, suggesting that the direct effect of type I IFNs on CD8 T cells is mediated by STAT1. Furthermore, effector CD8 T cells that lack STAT1 signaling did not survive the contraction phase to differentiate into long-lived memory cells. These results identify a critical role for type I IFN-STAT1 signaling in multiple stages of CD8 T cell response in vivo and suggest that strategies to activate type I IFN-STAT1 signaling pathway may enhance vaccine potency.
Leukemia Inhibitory Factor Deficiency Modulates the Immune Response and Limits Autoimmune Demyelination: A New Role for Neurotrophic Cytokines in Neuroinflammation
Authors: Linker Ralf A. , Kruse Niels , Israel Stephanie , Wei Tao , Seubert Silvia , Hombach Anja , Holtmann Bettina , Luhder Fred , Ransohoff Richard M. , Sendtner Michael , Gold Ralf
Published: April 2014

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF−/− mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF−/− mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF−/− mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-γ production in LIF−/− mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF−/− mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.
Transglutaminase Type II Is Involved in the Pathogenesis of Endotoxic Shock
Authors: Falasca Laura , Farrace Maria Grazia , Rinaldi Alessandra , Tuosto Loretta , Melino Gennard , Piacentini Mauro
Published: April 2014

The pathogenesis of sepsis is characterized by the inability of the host to regulate the inflammatory response, and as a consequence, dysregulated inflammatory processes induce organ dysfunctions and death. Altered transglutaminase type II (TG2) expression is associated with the development of many inflammatory diseases. Therefore, in this study, we questioned whether TG2 could also contribute to the pathological inflammatory dysregulation occurring in septic shock in vivo. To this aim, we used as an experimental model the TG2 knockout mice, in which the process of septic shock was elicited by treatment with LPS. Interestingly, our results demonstrated that TG2 ablation leads to partial resistance to experimental sepsis. The increased survival of TG2−/− mice was reflected in a drastic reduction of organ injury, highlighted by a limited infiltration of neutrophils in kidney and peritoneum and by a better homeostasis of the proinflammatory mediators as well as mitochondrial function. We also showed that in wild-type mice, the TG2 expression is increased during endotoxemia and, being directly involved in the mechanisms of NF-κB activation, it may cause a continuous activation cycle in the inflammatory process, thus contributing to development of sepsis pathogenesis. We propose that the inhibition of TG2 could represent a novel approach in the treatment of inflammatory processes associated with sepsis.
Cutting Edge: Vasoactive Intestinal Peptide (VIP) Induces Differentiation of Th17 Cells with a Distinctive Cytokine Profile
Authors: Yadav Mahesh , Rosenbaum Jennifer , Goetzl Edward J.
Published: April 2014

Immune cellular effects of vasoactive intestinal peptide (VIP) are transduced by VIP G protein-coupled receptors type 1 (VPAC1) and type 2 (VPAC2). We now show that VIP with TGFβ stimulates the transformation of CD4 T cells to a distinctive type of Th17 cell that generates IL-17 but not IL-6 or IL-21. VIP induction of Th17 cells was higher in VPAC2 knockout mice than wild-type mice, suggesting that VPAC1 is the principal transducer. Compared with Th17 cells elicited by IL-6, those evoked by VIP were similar in the secretion of IL-17 and IL-22, but lacked IL-21 secretion. Suppression of VIP induction of Th17 cells by protein kinase A inhibitors and enhancement by pharmacologically increased cAMP supports a role for this signal. The ability of VIP-VPAC1 axis signals to evoke development of a novel type of Th17 cells demonstrates the unique specificity of neuroregulatory mechanisms in the immunological environment.