bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Human CD36 in Bacterial Recognition, Phagocytosis, and Pathogen-Induced JNK-Mediated Signaling
Authors: Baranova Irina N. , Kurlander Roger , Bocharov Alexander V. , Vishnyakova Tatyana G. , Chen Zhigang , Remaley Alan T. , Csako Gyorgy , Patterson Amy P. , Eggerman Thomas L.
Published: April 2014

Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6 signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacterium, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus, and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both Gram-positive and Gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36 transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to Gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with Gram-positive bacteria and lipoteichoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40–50% in human fibroblasts induced by both Gram-negative and Gram-positive bacteria as well as LPS. Of all MAPK signaling cascade inhibitors tested, only the inhibitor of JNK, a stress-activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-κB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an α-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by Gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner.
NOD2-Deficient Mice Have Impaired Resistance to Mycobacterium tuberculosis Infection through Defective Innate and Adaptive Immunity
Authors: Divangahi Maziar , Mostowy Serge , Coulombe François , Kozak Robert , Guillot Loïc , Veyrier Frédéric , Kobayashi Koichi S. , Flavell Richard A. , Gros Philippe , Behr Marcel A.
Published: April 2014

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2−/− mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2−/− mice had decreased production of type 1 cytokines and reduced recruitment of CD8+ and CD4+ T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2−/− mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2−/− mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.
The ESX-5 Secretion System of Mycobacterium marinum Modulates the Macrophage Response
Authors: Abdallah Abdallah M. , Savage Nigel D. L. , van Zon Maaike , Wilson Louis , Vandenbroucke-Grauls Christina M. J. E. , van der Wel Nicole N. , Ottenhoff Tom H. M. , Bitter Wilbert
Published: April 2014

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mφ). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-α, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1β production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mφ. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mφ response at various critical steps.
TLR/MyD88 and Liver X Receptor α Signaling Pathways Reciprocally Control Chlamydia pneumoniae-Induced Acceleration of Atherosclerosis
Authors: Naiki Yoshikazu , Sorrentino Rosalinda , Wong Michelle H. , Michelsen Kathrin S. , Shimada Kenichi , Chen Shuang , Yilmaz Atilla , Slepenkin Anatoly , Schröder Nicolas W. J. , Crother Timothy R. , Bulut Yonca , Doherty Terence M. , Bradley Michelle , Shaposhnik Zory , Peterson Ellena M. , Tontonoz Peter , Shah Prediman K. , Arditi Moshe
Published: April 2014

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)−/− mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor α (LXRα) signaling pathways. We infected ApoE−/− mice and ApoE−/− mice that also lacked TLR2, TLR4, MyD88, or LXRα intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-α. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE−/− mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE−/− mice was further enhanced in ApoE−/−LXRα−/− double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-α. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRα appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.
Chlamydia pneumoniae-Induced Foam Cell Formation Requires MyD88-Dependent and -Independent Signaling and Is Reciprocally Modulated by Liver X Receptor Activation
Authors: Chen Shuang , Sorrentino Rosalinda , Shimada Kenichi , Bulut Yonca , Doherty Terence M. , Crother Timothy R. , Arditi Moshe
Published: April 2014

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.
Macrophage Antigen Complex-1 Mediates Reactive Microgliosis and Progressive Dopaminergic Neurodegeneration in the MPTP Model of Parkinson’s Disease
Authors: Hu Xiaoming , Zhang Dan , Pang Hao , Caudle W. Michael , Li Yachen , Gao Huiming , Liu Yuxin , Qian Li , Wilson Belinda , Di Monte Donato A. , Ali Syed F. , Zhang Jing , Block Michelle L. , Hong Jau-Shyong
Published: April 2014

Neuronal death is known to trigger reactive microgliosis. However, little is known regarding the manner by which microglia are activated by injured neurons and how microgliosis participates in neurodegeneration. In this study we delineate the critical role of macrophage Ag complex-1 (MAC1), a member of the β2 integrin family, in mediating reactive microgliosis and promoting dopaminergic (DAergic) neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. MAC1 deficiency greatly attenuated the DAergic neurodegeneration induced by MPTP or 1-methyl-4-phenyl-pyridium iodide (MPP+) exposure both in vivo and in vitro, respectively. Reconstituted experiments created by adding microglia from MAC1−/− or MAC1+/+ mice back to MAC1+/+ neuron-enriched cultures showed that microglia with functional MAC1 expression was mandatory for microglia-enhanced neurotoxicity. Both in vivo and in vitro morphological and Western blot studies demonstrated that MPTP/MPP+ produced less microglia activation in MAC1−/− mice than MAC1+/+ mice. Further mechanistic studies revealed that a MPP+-mediated increase in superoxide production was reduced in MAC1−/− neuron-glia cultures compared with MAC1+/+ cultures. The stunted production of superoxide in MAC1−/− microglia is likely linked to the lack of translocation of the cytosolic NADPH oxidase (PHOX) subunit (p47phox) to the membrane. In addition, the production of PGE2 markedly decreased in neuron plus MAC1−/− microglia cocultures vs neuron plus MAC1+/+ microglia cocultures. Taken together, these results demonstrate that MAC1 plays a critical role in MPTP/MPP+-induced reactive microgliosis and further support the hypothesis that reactive microgliosis is an essential step in the self-perpetuating cycle leading to progressive DAergic neurodegeneration observed in Parkinson’s disease.
CD4+CD25+ Foxp3+ Regulatory T Cells Protect against T Cell-Mediated Fulminant Hepatitis in a TGF-β-Dependent Manner in Mice
Authors: Wei Hua-Xing , Chuang Ya-Hui , Li Bofeng , Wei Haiming , Sun Rui , Moritoki Yuki , Gershwin M. Eric , Lian Zhe-Xiong , Tian Zhigang
Published: April 2014

Regulatory T cells (Tregs), which are characterized by expression of CD4, CD25, and Foxp3, play a crucial role in the control of immune responses to both self and non-self Ags. To date, there are only limited data on their role in physiological and pathological hepatic immune responses. In this study, we examined the role of hepatic Tregs in immune-mediated liver injury by using the murine Con A-induced hepatitis model. Con A treatment was associated with an increased number of Foxp3+ Tregs in liver but not in spleen. Moreover, the expression levels of Foxp3, CTLA-4, glucocorticoid-induced TNF receptor, as well as the frequency of CD103 of Tregs were increased after Con A injection, being significantly higher in liver than in spleen. Depleting CD25+ cells aggravated liver injury, whereas adoptively transferring CD25+ cells or Tregs reduced liver injury in Con A-treated recipients. Con A treatment induced elevated serum levels and hepatic mononuclear mRNA expressions of TGF-β, which were reduced by Tregs depletion. In addition, anti-TGF-β mAbs blocked the suppressive function of Tregs from Con A-treated mice in vitro. Finally, TGF-β receptor II dominant-negative mice, whose T cells express a dominant negative form of TGFβRII and therefore cannot respond to TGF-β, had a higher mortality rate and severer liver injury than normal mice injected with the same dose of Con A. These results indicate that CD4+CD25+ Tregs play an important role in limiting the liver injury in Con A-induced hepatitis via a TGF-β-dependent mechanism.
A New Mechanism for Inhalational Priming: IL-4 Bypasses Innate Immune Signals
Authors: Dittrich Anna M. , Chen Hui-Chen , Xu Lan , Ranney Patricia , Connolly Sean , Yarovinsky Timur O. , Bottomly H. Kim
Published: April 2014

Signaling via innate immune mechanisms is considered pivotal for T cell-mediated responses to inhaled Ags. Furthermore, Th2 cells specific for one inhaled Ag can facilitate priming of naive T cells to unrelated new inhaled Ags, a process we call “Th2 collateral priming”. Interestingly, our previous studies showed that collateral priming is independent of signals via the innate immune system but depends on IL-4 secretion by CD4+ T cells. We thus hypothesized that IL-4 can bypass the need for signals via the innate immune system, considered essential for pulmonary priming. Indeed, we were able to show that IL-4 bypasses the requirement for TLR4- and MyD88-mediated signaling for responses to new allergens. Furthermore, we characterized the mechanisms by which IL-4 primes for new inhaled allergens: “IL-4-dependent pulmonary priming” relies on IL-4 receptor expression on hematopoietic cells and structural cells. Transfer experiments indicate that within the hematopoietic compartment both T cells and dendritic cells need to express the IL-4 receptor. Finally, we were able to show that IL-4 induces recruitment and maturation of myeloid dendritic cells in vivo and increases T cell recruitment to the draining lymph nodes. Our findings bring new mechanistic knowledge to the phenomenon of polysensitization and primary sensitization in asthma.
Dual Regulatory Roles of Phosphatidylinositol 3-Kinase in IFN Signaling
Authors: Kaur Surinder , Sassano Antonella , Joseph Ajith M. , Majchrzak-Kita Beata , Eklund Elizabeth A. , Verma Amit , Brachmann Saskia M. , Fish Eleanor N. , Platanias Leonidas C.
Published: April 2014

PI3K is activated by the type I and II IFN receptors, but its precise role in the generation of IFN responses is not well understood. In the present study we used embryonic fibroblasts from mice with targeted disruption of the genes encoding for both the p85α and p85β regulatory subunits of PI3′-kinase (p85α−/−β−/−) to precisely define the role of PI3K in the control of IFN-induced biological responses. Our data demonstrate that PI3K plays dual regulatory roles in the induction of IFN responses by controlling both IFN-α- and IFN-γ-dependent transcriptional regulation of IFN-sensitive genes and simultaneously regulating the subsequent initiation of mRNA translation for such genes. These processes include the Isg15, Cxcl10, and/or Irf7 genes, whose functions are important in the generation of the biological effects of IFNs. Consistent with this, the induction of IFN antiviral responses is defective in double p85α/p85β knockout cells. Thus, integration of signals via PI3K is a critical event during engagement of the IFN receptors that complements both the transcriptional activity of Jak-STAT pathways and controls initiation of mRNA translation.
Induction of Scavenger Receptor Class B Type I Is Critical for Simvastatin Enhancement of High-Density Lipoprotein-Induced Anti-Inflammatory Actions in Endothelial Cells
Authors: Kimura Takao , Mogi Chihiro , Tomura Hideaki , Kuwabara Atsushi , Im Doon-Soon , Sato Koichi , Kurose Hitoshi , Murakami Masami , Okajima Fumikazu
Published: April 2014

Changes in plasma lipoprotein profiles, especially low levels of high-density lipoprotein (HDL), are a common biomarker for several inflammatory and immune diseases, including atherosclerosis and rheumatoid arthritis. We examined the effect of simvastatin on HDL-induced anti-inflammatory actions. HDL and sphingosine 1-phosphate (S1P), a bioactive lipid component of the lipoprotein, inhibited TNF α-induced expression of VCAM-1, which was associated with NO synthase (NOS) activation, in human umbilical venous endothelial cells. The HDL- but not S1P-induced anti-inflammatory actions were enhanced by a prior treatment of the cells with simvastatin in a manner sensitive to mevalonic acid. Simvastatin stimulated the expression of scavenger receptor class B type I (SR-BI) and endothelial NOS. As for S1P receptors, however, the statin inhibited the expression of S1P3 receptor mRNA but caused no detectable change in S1P1 receptor expression. The reconstituted HDL, a stimulator of SR-BI, mimicked HDL actions in a simvastatin-sensitive manner. The HDL- and reconstituted HDL-induced actions were blocked by small interfering RNA specific to SR-BI regardless of simvastatin treatment. The statin-induced expression of SR-BI was attenuated by constitutively active RhoA and small interfering RNA specific to peroxisome proliferator-activated receptor-α. Administration of simvastatin in vivo stimulated endothelial SR-BI expression, which was accompanied by the inhibition of the ex vivo monocyte adhesion in aortas from TNF α-injected mice. In conclusion, simvastatin induces endothelial SR-BI expression through a RhoA- and peroxisome proliferator-activated receptor-α-dependent mechanism, thereby enhancing the HDL-induced activation of NOS and the inhibition of adhesion molecule expression.
Intestinal Macrophage/Epithelial Cell-Derived CCL11/Eotaxin-1 Mediates Eosinophil Recruitment and Function in Pediatric Ulcerative Colitis
Authors: Ahrens Richard , Waddell Amanda , Seidu Luqman , Blanchard Carine , Carey Rebecca , Forbes Elizabeth , Lampinen Maria , Wilson Tara , Cohen Elizabeth , Stringer Keith , Ballard Edgar , Munitz Ariel , Xu Huan , Lee Nancy , Lee James J. , Rothenberg Marc E. , Denson Lee , Hogan Simon P.
Published: April 2014

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2−/− and eotaxin-1/2−/− mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80+CD11b+ macrophages in DSS-induced epithelial injury and to CD68+ intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.
Multiple Glycines in TCR α-Chains Determine Clonally Diverse Nature of Human T Cell Memory to Influenza A Virus
Authors: Naumov Yuri N. , Naumova Elena N. , Yassai Maryam B. , Kota Kalyani , Welsh Raymond M. , Selin Liisa K.
Published: April 2014

Detailed assessment of how the structural properties of T cell receptors affect clonal repertoires of Ag-specific cells is a prerequisite for a better understanding of human antiviral immunity. Herein we examine the α TCR repertoires of CD8 T cells reactive against the influenza A viral epitope M158–66, restricted by HLA-A2.1. Using molecular cloning, we systematically studied the impact of α-chain usage in the formation of T cell memory and revealed that M158–66-specific, clonally diverse VB19 T cells express α-chains encoded by multiple AV genes with different CDR3 sizes. A unique feature of these α TCRs was the presence of CDR3 fitting to an AGA(Gn)GG-like amino acid motif. This pattern was consistent over time and among different individuals. Further molecular assessment of human CD4+CD8− and CD4−CD8+ thymocytes led to the conclusion that the poly-Gly/Ala runs in CDR3α were a property of immune, but not naive, repertoires and could be attributed to influenza exposure. Repertoires of T cell memory are discussed in the context of clonal diversity, where poly-Gly/Ala runs in the CDR3 of α- and β-chains might provide high levels of TCR flexibility during Ag recognition while gene-encoded CDR1 and CDR2 contribute to the fine specificity of the TCR-peptide MHC interaction.
Low Expression of the IL-23/Th17 Pathway in Atopic Dermatitis Compared to Psoriasis
Authors: Guttman-Yassky Emma , Lowes Michelle A. , Fuentes-Duculan Judilyn , Zaba Lisa C. , Cardinale Irma , Nograles Kristine E. , Khatcherian Artemis , Novitskaya Inna , Carucci John A. , Bergman Reuven , Krueger James G.
Published: April 2014

The classical Th1/Th2 paradigm previously defining atopic dermatitis (AD) and psoriasis has recently been challenged with the discovery of Th17 T cells that synthesize IL-17 and IL-22. Although it is becoming evident that many Th1 diseases including psoriasis have a strong IL-17 signal, the importance of Th17 T cells in AD is still unclear. We examined and compared skin biopsies from AD and psoriasis patients by gene microarray, RT-PCR, immunohistochemistry, and immunofluorescence. We found a reduced genomic expression of IL-23, IL-17, and IFN-γ in AD compared with psoriasis. To define the effects of IL-17 and IL-22 on keratinocytes, we performed gene array studies with cytokine-treated keratinocytes. We found lipocalin 2 and numerous other innate defense genes to be selectively induced in keratinocytes by IL-17. IFN-γ had no effect on antimicrobial gene-expression in keratinocytes. In AD skin lesions, protein and mRNA expression of lipocalin 2 and other innate defense genes (hBD2, elafin, LL37) were reduced compared with psoriasis. Although AD has been framed by the Th1/Th2 paradigm as a Th2 polar disease, we present evidence that the IL-23/Th17 axis is largely absent, perhaps accounting for recurrent skin infections in this disease.
Cutting Edge: TheIdd3Genetic Interval Determines Regulatory T Cell Function through CD11b+CD11c− APC
Authors: Anderson Ana C. , Chandwaskar Rucha , Lee David H. , Kuchroo Vijay K.
Published: April 2014

The Idd3 genetic interval confers protection against multiple autoimmune diseases, including type 1 diabetes and experimental autoimmune encephalomyelitis. The favored candidate gene in this interval is Il2, which is polymorphic between susceptible and resistant strains of mice. IL-2 regulates the growth/death of effector T cells as well as the generation/maintenance of regulatory T cells (Tregs), and recent studies have shown that NOD.Idd3 Tregs are more suppressive than their NOD counterparts. We have further dissected the mechanisms underlying the differential suppression by NOD and NOD.Idd3 Tregs and find that it is determined by CD11b+CD11c− APCs. Thus, contrary to what might be expected, our data suggest that the differential suppressive activity of NOD and NOD.Idd3 Tregs is not due to an effect of the Idd3 genetic interval on T cells but rather is due to differences in the APC compartment.
Increased Osteopontin Expression in Dendritic Cells Amplifies IL-17 Production by CD4+ T Cells in Experimental Autoimmune Encephalomyelitis and in Multiple Sclerosis
Authors: Murugaiyan Gopal , Mittal Akanksha , Weiner Howard L.
Published: April 2014

Osteopontin (Opn) is a broadly expressed pleiotropic cytokine, and has been shown to play an important role in various autoimmune diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). It is reported that Opn exacerbates EAE by skewing T cell differentiation toward IFN-γ-producing Th1 cells. Opn expression in dendritic cells (DCs) and its role in IL-17 induction from T cells during EAE or MS are unknown. We found that during EAE, Opn expression is elevated in DCs both in the periphery and in the CNS. There was increased expression of Opn receptor on T cells, and Opn induced IL-17 production by CD4+ T cells via the β3 integrin receptor and Opn inhibited IL-10 production via the CD44 receptor. Furthermore, anti-Opn treatment reduced clinical severity of EAE by reducing IL-17 production. Anti-Opn was also effective in reducing clinical severity of EAE when given after the appearance of clinical symptoms. Analogous to EAE, in subjects with MS, we found increased expression of Opn in DCs and increased expression of the Opn receptors CD44, β3, and αv on T cells. Furthermore, Opn-stimulated CD4+ T cells from MS patients produced significantly higher amounts of IL-17. Our results demonstrate a role for DC-produced Opn both in EAE and MS that is linked to the production of IL-17.
NK Cells Induce Apoptosis in Tubular Epithelial Cells and Contribute to Renal Ischemia-Reperfusion Injury
Authors: Zhang Zhu-Xu , Wang Shuang , Huang Xuyan , Min Wei-Ping , Sun Hongtao , Liu Weihua , Garcia Bertha , Jevnikar Anthony M.
Published: April 2014

Renal ischemia-reperfusion injury (IRI) can result in acute renal failure with mortality rates of 50% in severe cases. NK cells are important participants in early-stage innate immune responses. However, their role in renal tubular epithelial cell (TEC) injury in IRI is currently unknown. Our data indicate that NK cells can kill syngeneic TEC in vitro. Apoptotic death of TEC in vitro is associated with TEC expression of the NK cell ligand Rae-1, as well as NKG2D on NK cells. In vivo following IRI, there was increased expression of Rae-1 on TEC. FACS analyses of kidney cell preparations indicated a quantitative increase in NKG2D-bearing NK cells within the kidney following IRI. NK cell depletion in wild-type C57BL/6 mice was protective, while adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2−/−γc−/− mice with IRI. NK cell-mediated kidney injury was perforin (PFN)-dependent as PFN−/− NK cells had minimal capacity to kill TEC in vitro compared with NK cells from wild-type, FasL-deficient (gld), or IFN-γ−/− mice. Taken together, these results demonstrate for the first time that NK cells can directly kill TEC and that NK cells contribute substantially to kidney IRI. NK cell killing may represent an important underrecognized mechanism of kidney injury in diverse forms of inflammation, including transplantation.
Inhibition of the Alloimmune Response through the Generation of Regulatory T Cells by a MHC Class II-Derived Peptide
Authors: Zang Weiping , Lin Marvin , Kalache Safa , Zhang Nan , Krüger Bernd , Waaga-Gasser Ana Maria , Grimm Martin , Hancock Wayne , Heeger Peter , Schröppel Bernd , Murphy Barbara
Published: April 2014

We have previously shown that HLA-DQA1, a peptide derived from a highly conserved region of MHC class II, prevents alloreactive T cell priming and effector function in vivo, although underlying mechanisms are obscure. In this study, we demonstrate that 28% of mice treated with HLA-DQA1 combined with low-dose rapamycin achieved permanent engraftment of fully MHC-disparate islet allografts and significantly prolonged survival in the remaining animals (log rank, p < 0.001). Immunohistologic examination of the grafts from HLA-DQA1/rapamycin-treated animals revealed up-regulated expression of TGF-ß and FoxP3. In vivo administration of blocking anti-TGF-ß or depleting anti-CD25 mAb augmented T cell alloimmunity and prevented the long-term engraft induced by HLA-DQA1. In vitro experiments further showed that HLA-DQA1 induced differentiation of CD4+ T cells into CD4+CD25+FoxP3+ regulatory T cells. Together, these data provide the first demonstration that HLA-DQA1, a MHC class II-derived peptide, can prolong allograft survival via a TGF-β and regulatory T cell-dependent mechanisms.
Resolution of Unique Sca-1highc-Kit− Lymphoid-Biased Progenitors in Adult Bone Marrow
Authors: Harman Benjamin C. , Northrup Daniel L. , Allman David
Published: April 2014

We have identified a distinctive lymphoid-restricted progenitor population in adult mouse bone marrow based on a unique c-Kit−Sca-1highFlt3+ AA4+ surface phenotype. These cells are highly lymphoid biased and rapidly generate B and T cells after adoptive transfer. However, whereas previously described lymphoid progenitors such as common lymphoid progenitors express TdT and relatively high levels of RAG2, and are enriched for cells with an active V(D)J recombinase, Flt3+ AA4+ cells within the c-Kit−Sca-1high bone marrow fraction are TdT−, are RAG2low, and do not display evidence for ongoing or past recombinase activity. Furthermore, unlike common lymphoid progenitors that readily generate B cells upon stimulation with IL-7, c-Kit−Sca-1highFlt3+ precursors do not express abundant levels of the IL-7R, and require costimulation with Flt3 ligand and IL-7 to generate B cells in vitro. Moreover, these findings suggest that hematopoietic stem cells in adults generate an array of lymphoid-biased progenitor populations characterized by distinct gene expression and cytokine response profiles.
Retinal Pigment Epithelium-Derived CTLA-2α Induces TGFβ-Producing T Regulatory Cells
Authors: Sugita Sunao , Horie Shintaro , Nakamura Orie , Futagami Yuri , Takase Hiroshi , Keino Hiroshi , Aburatani Hiroyuki , Katunuma Nobuhiko , Ishidoh Kazumi , Yamamoto Yoshimi , Mochizuki Manabu
Published: April 2014

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4+ T cells into Treg cells by producing and secreting CTLA-2α, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2α converted CD4+ T cells into Treg cells in vitro, and CTLA-2α small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2α induced CD4+CD25+Foxp3+ Treg cells that produced TGFβ in vitro. Moreover, CTLA-2α produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4+ T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2α effect on Treg cell induction occurred through TGFβ signaling, because CTLA-2α promoted activation of TGFβ in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.
Targeting the Neonatal Fc Receptor for Antigen Delivery Using Engineered Fc Fragments
Authors: Mi Wentao , Wanjie Sylvia , Lo Su-Tang , Gan Zhuo , Pickl-Herk Beatrix , Ober Raimund J. , Ward E. Sally
Published: April 2014

The development of approaches for Ag delivery to the appropriate subcellular compartments of APCs and the optimization of Ag persistence are both of central relevance for the induction of protective immunity or tolerance. The expression of the neonatal Fc receptor, FcRn, in APCs and its localization to the endosomal system suggest that it might serve as a target for Ag delivery using engineered Fc fragment-epitope fusions. The impact of FcRn binding characteristics of an Fc fragment on in vivo persistence allows this property to also be modulated. We have therefore generated recombinant Fc (mouse IgG1-derived) fusions containing the N-terminal epitope of myelin basic protein that is associated with experimental autoimmune encephalomyelitis in H-2u mice. The Fc fragments have distinct binding properties for FcRn that result in differences in intracellular trafficking and in vivo half-lives, allowing the impact of these characteristics on CD4+ T cell responses to be evaluated. To dissect the relative roles of FcRn and the “classical” FcγRs in Ag delivery, analogous aglycosylated Fc-MBP fusions have been generated. We show that engineered Fc fragments with increased affinities for FcRn at pH 6.0–7.4 are more effective in delivering Ag to FcRn-expressing APCs in vitro relative to their lower affinity counterparts. However, higher affinity of the FcRn-Fc interaction at near neutral pH results in decreased in vivo persistence. The trade-off between improved FcRn targeting efficiency and lower half-life becomes apparent during analyses of T cell proliferative responses in mice, particularly when Fc-MBP fusions with both FcRn and FcγR binding activity are used.
Transplantation of Bone Marrow Transduced to Express Self-Antigen Establishes Deletional Tolerance and Permanently Remits Autoimmune Disease
Authors: Chan James , Ban Ee Jun , Chun Keng Hao , Wang Shunhe , Bäckström B. Thomas , Bernard Claude C. A. , Toh Ban-Hock , Alderuccio Frank
Published: April 2014

Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-γ, and GM-CSF in response to MOG35–55 peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4+ MOG35–55-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.
Drak2 Regulates the Survival of Activated T Cells and Is Required for Organ-Specific Autoimmune Disease
Authors: McGargill Maureen A. , Choy Carmen , Wen Ben G. , Hedrick Stephen M.
Published: April 2014

Drak2 is a serine/threonine kinase expressed in T and B cells. The absence of Drak2 renders T cells hypersensitive to suboptimal stimulation, yet Drak2−/− mice are enigmatically resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We show in this study that Drak2−/− mice were also completely resistant to type 1 diabetes when bred to the NOD strain of mice that spontaneously develop autoimmune diabetes. However, there was not a generalized suppression of the immune system, because Drak2−/− mice remained susceptible to other models of autoimmunity. Adoptive transfer experiments revealed that resistance to disease was intrinsic to the T cells and was due to a loss of T cell survival under conditions of chronic autoimmune stimulation. Importantly, the absence of Drak2 did not alter the survival of naive T cells, memory T cells, or T cells responding to an acute viral infection. These experiments reveal a distinction between the immune response to persistent self-encoded molecules and transiently present infectious agents. We present a model whereby T cell survival depends on a balance of TCR and costimulatory signals to explain how the absence of Drak2 affects autoimmune disease without generalized suppression of the immune system.
Enhanced T Cell Apoptosis withinDrak2-Deficient Mice Promotes Resistance to Autoimmunity
Authors: Ramos Stephanie J. , Hernandez Jeniffer B. , Gatzka Martina , Walsh Craig M.
Published: April 2014

Clonal expansion of T cells is vital to adaptive immunity, yet this process must be tightly controlled to prevent autoimmune disease. The serine/threonine kinase death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK2) is a negative regulator of TCR signaling and sets the threshold for the activation of naive and memory T cells and selected thymocytes. Despite enhanced T cell activation, Drak2−/− mice are resistant to experimental autoimmune encephalomyelitis, an autoimmune demyelinating disease that resembles multiple sclerosis. However, the basis for this autoimmune resistance is currently unknown. In this study, we show that, in the absence of DRAK2 signaling, T cells require greater tonic signaling for maintenance during clonal expansion. Following stimulation, Drak2−/− T cells were more sensitive to an intrinsic form of apoptosis that was prevented by CD28 ligation, homeostatic cytokines, or enforced Bcl-xL expression. T cell-specific Bcl-xL expression also restored the susceptibility of Drak2−/− mice to experimental autoimmune encephalomyelitis and enhanced thymic positive selection. These findings demonstrate that DRAK2 is selectively important for T cell survival and highlight the potential that DRAK2 blockade may lead to permanent autoimmune T cell destruction via intrinsic apoptosis pathways.
The Erk2 MAPK Regulates CD8 T Cell Proliferation and Survival
Authors: D'Souza Warren N. , Chang Chiung-Fang , Fischer April M. , Li Manqing , Hedrick Stephen M.
Published: April 2014

The magnitude of T cell responses is determined by proliferation and survival decisions made by the responding cells. We now demonstrate that the Erk MAPK pathway plays a critical role in these cell fate decisions within CD8 T cells. While Erk1 is dispensable for all aspects of CD8 T cell activation, Erk2 is required for the proliferation of CD8 T cells activated in the absence of costimulation. Surprisingly, Erk2 is not required for proliferation following the addition of a costimulatory signal in vitro, or upon viral infection in vivo, but regulates the size of the responding population by enhancing cell survival. An important component of this Erk2-derived signal is the transcriptional regulation of Bcl-2 family members Bcl-xL and Bim, and impaired Erk2-deficient CD8 T cell survival can be rescued by genetic ablation of Bim. These studies ascribe multifaceted functions specific to Erk2 in CD8 T cell activation, proliferation, and survival.
Targeting Poly(I:C) to the TLR3-Independent Pathway Boosts Effector CD8 T Cell Differentiation through IFN-α/β
Authors: Ngoi Soo M. , Tovey Michael G. , Vella Anthony T.
Published: April 2014

Poly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3−/− mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3−/− mice. A higher percentage of Ag-specific CD8 T cells became IFN-γ and TNF-α producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-α/β-dependent. Administration of IFN-α/β-neutralizing Abs abolished the poly(I:C) effects in TLR3−/− mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.
Dynamic Modulation of CCR7 Expression and Function on Naive T Lymphocytes In Vivo
Authors: Britschgi Mirjam R. , Link Alexander , Lissandrin Tonje Katrine A. , Luther Sanjiv A.
Published: April 2014

The chemokine receptor CCR7 is critical for the recirculation of naive T cells. It is required for T cell entry into secondary lymphoid organs (SLO) and for T cell motility and retention within these organs. How CCR7 activity is regulated during these processes in vivo is poorly understood. Here we show strong modulation of CCR7 surface expression and occupancy by the two CCR7 ligands, both in vitro and in vivo. In contrast to blood, T cells in SLO had most surface CCR7 occupied with CCL19, presumably leading to continuous signaling and cell motility. Both ligands triggered CCR7 internalization in vivo as shown in Ccl19−/− and plt/plt mice. Importantly, CCR7 occupancy and down-regulation led to strongly impaired chemotactic responses, an effect reversible by CCR7 resensitization. Therefore, during their recirculation, T cells cycle between states of free CCR7 with high ligand sensitivity in blood and occupied CCR7 associated with continual signaling and reduced ligand sensitivity within SLO. We propose that these two states of CCR7 are important to allow the various functions CCR7 plays in T cell recirculation.
Retinoic Acids Are Potent Inhibitors of Spontaneous Human Eosinophil Apoptosis
Authors: Ueki Shigeharu , Mahemuti Gulixian , Oyamada Hajime , Kato Hikari , Kihara Junko , Tanabe Masako , Ito Wataru , Chiba Takahito , Takeda Masahide , Kayaba Hiroyuki , Chihara Junichi
Published: April 2014

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10−6M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.
The Role of Thymic Stromal Lymphopoietin in CD8+ T Cell Homeostasis
Authors: Rochman Yrina , Leonard Warren J.
Published: April 2014

Thymic stromal lymphopoietin (TSLP) is a cytokine produced by stromal cells, epithelial cells, and basophils that acts on dendritic cells, mast cells, and CD4+ T cells. The receptor for TSLP contains a TSLP-specific receptor chain (TSLPR) and the IL-7R α-chain. Although IL-7 critically controls the expansion and survival of naive and memory CD8+ T cells, an action for TSLP on CD8+ T cells has not been reported. We now demonstrate that CD8+ T cells express TSLPR and that TSLP activates both STAT5 and Akt and induces Bcl-2 in these cells. Correspondingly, TSLP increases CD8+ T cell survival in vitro as well as in wild-type and T-depleted mice in vivo, without altering the homeostatic proliferation of these cells. Moreover, TSLP can maintain CD8+ T cells even in the absence of IL-7. Thus, our data reveal that TSLP contributes to CD8+ T cell homeostasis in both normal and lymphopenic conditions.
The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells
Authors: Kuehn Hye Sun , Swindle Emily J. , Kim Mi-Sun , Beaven Michael A. , Metcalfe Dean D. , Gilfillan Alasdair M.
Published: April 2014

Activated mast cells are a major source of the eicosanoids PGD2 and leukotriene C4 (LTC4), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A2, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC4 and PGD2, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcεRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcεRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcεRI-dependent PGD2, LTC4, and ROS production in bone marrow-derived mast cells of Btk−/− mice. The FcεRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A2 phosphorylation but rather appears to regulate the generation of ROS, LTC4, and PGD2 by contributing to the necessary Ca2+ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.