Journal of Experimental Medicine recent issues

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Mon, 26 Oct 2009 09:19:01 PDT

Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis

Mon, 26 Oct 2009 09:19:01 PDT

Mast cells' message in a particle

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

Revising the Th17 recipe

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

How staph thwarts attack

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

Runx: T reg cell keeper and creator

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

Pro-fibrotic SNPs

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

Salim "Slim" Abdool Karim: Attacking AIDS in South Africa

Maxmen, A. — Mon, 26 Oct 2009 09:18:59 PDT

Masking MALT1: the paracaspase's potential for cancer therapy

Vucic, D., Dixit, V. M. — Mon, 26 Oct 2009 09:19:00 PDT

A key feature of aggressive B cell lymphomas is constitutive NF-B activation, which requires signals from the CARD11–BCL-10–MALT1 (CMB) complex. The unique enzymatic activity of MALT1 degrades one of its binding partners, BCL-10, as well as the NF-B inhibitor A20. New data shows that targeting MALT1 protease activity may be a promising therapeutic strategy for treating aggressive B cell lymphomas.

Inhibition of MALT1 protease activity is selectively toxic for activated B cell-like diffuse large B cell lymphoma cells

Mon, 26 Oct 2009 09:19:00 PDT

Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell–like (ABC) subtype of DLBCL is characterized by constitutive NF-B activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11–BCL10–MALT1 (CBM) complexes couple upstream events to IB kinase (IKK)/NF-B activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-B regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell–like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-B activity, and decreases the expression of NF-B targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.

Variants of CTGF are associated with hepatic fibrosis in Chinese, Sudanese, and Brazilians infected with Schistosomes

Mon, 26 Oct 2009 09:19:00 PDT

Abnormal fibrosis occurs during chronic hepatic inflammations and is the principal cause of death in hepatitis C virus and schistosome infections. Hepatic fibrosis (HF) may develop either slowly or rapidly in schistosome-infected subjects. This depends, in part, on a major genetic control exerted by genes of chromosome 6q23. A gene (connective tissue growth factor [CTGF]) is located in that region that encodes a strongly fibrogenic molecule. We show that the single nucleotide polymorphism (SNP) rs9402373 that lies close to CTGF is associated with severe HF (P = 2 x 10^–6; odds ratio [OR] = 2.01; confidence interval of OR [CI] = 1.51–2.7) in two Chinese samples, in Sudanese, and in Brazilians infected with either Schistosoma japonicum or S. mansoni. Furthermore, SNP rs12526196, also located close to CTGF, is independently associated with severe fibrosis (P = 6 x 10^–4; OR = 1.94; CI = 1.32–2.82) in the Chinese and Sudanese subjects. Both variants affect nuclear factor binding and may alter gene transcription or transcript stability. The identified variants may be valuable markers for the prediction of disease progression, and identify a critical step in the development of HF that could be a target for chemotherapy.

Runx proteins regulate Foxp3 expression

Mon, 26 Oct 2009 09:19:00 PDT

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice

Mon, 26 Oct 2009 09:19:00 PDT

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c⁺ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11c^hi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.

Id2-, ROR{gamma}t-, and LT{beta}R-independent initiation of lymphoid organogenesis in ocular immunity

Mon, 26 Oct 2009 09:19:00 PDT

The eye is protected by the ocular immunosurveillance system. We show that tear duct–associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid–related orphan receptor t, lymphotoxin (LT) 1β2–LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo

Mon, 26 Oct 2009 09:19:00 PDT

Platelet activation at a site of vascular injury is essential for the arrest of bleeding; however, excessive platelet activation at a site of arterial damage can result in the unwarranted formation of arterial thrombi, precipitating acute myocardial infarction, or ischemic stroke. Activation of platelets beyond the purpose of hemostasis may occur when substances facilitating thrombus growth and stability accumulate. Human platelets contain matrix metalloproteinase 2 (MMP-2) and release it upon activation. Active MMP-2 amplifies the platelet aggregation response to several agonists by potentiating phosphatidylinositol 3-kinase activation. Using several in vivo thrombosis models, we show that the inactivation of the MMP-2 gene prevented thrombosis induced by weak, but not strong, stimuli in mice but produced only a moderate prolongation of the bleeding time. Moreover, using cross-transfusion experiments and wild-type/MMP-2^–/– chimeric mice, we show that it is platelet-derived MMP-2 that facilitates thrombus formation. Finally, we show that platelets activated by a mild vascular damage induce thrombus formation at a downstream arterial injury site by releasing MMP-2. Thus, platelet-derived MMP-2 plays a crucial role in thrombus formation by amplifying the response of platelets to weak activating stimuli. These findings open new possibilities for the prevention of thrombosis by the development of MMP-2 inhibitors.

Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis

Mon, 26 Oct 2009 09:19:00 PDT

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Dependence of proliferative vascular smooth muscle cells on CD98hc (4F2hc, SLC3A2)

Fogelstrand, P., Feral, C. C., Zargham, R., Ginsberg, M. H. — Mon, 26 Oct 2009 09:19:00 PDT

Activation of vascular smooth muscle cells (VSMCs) to migrate and proliferate is essential for the formation of intimal hyperplasia. Hence, selectively targeting activated VSMCs is a potential strategy against vaso-occlusive disorders such as in-stent restenosis, vein-graft stenosis, and transplant vasculopathy. We show that CD98 heavy chain (CD98hc) is markedly up-regulated in neointimal and cultured VSMCs, and that activated but not quiescent VSMCs require CD98hc for survival. CD98hc mediates integrin signaling and localizes amino acid transporters to the plasma membrane. SMC-specific deletion of CD98hc did not affect normal vessel morphology, indicating that CD98hc was not required for the maintenance of resident quiescent VSMCs; however, CD98hc deletion reduced intimal hyperplasia after arterial injury. Ex vivo and in vitro, loss of CD98hc suppressed proliferation and induced apoptosis in VSMCs. Furthermore, reconstitution with CD98hc mutants showed that CD98hc interaction with integrins was necessary for the survival of VSMCs. These studies establish the importance of CD98hc in VSMC proliferation and survival. Furthermore, loss of CD98hc was selectively deleterious to activated VSMCs while sparing resident quiescent VSMCs, suggesting that activated VSMCs are physiologically dependent on CD98hc, and hence, CD98hc is a potential therapeutic target in vaso-occlusive disorders.

Transforming growth factor {beta} is dispensable for the molecular orchestration of Th17 cell differentiation

Mon, 26 Oct 2009 09:19:00 PDT

Interleukin (IL)-17–producing T helper (Th17) cells play a critical role in the pathophysiology of several autoimmune disorders. The differentiation of Th17 cells requires the simultaneous presence of an unusual combination of cytokines: IL-6, a proinflammatory cytokine, and transforming growth factor (TGF) β, an antiinflammatory cytokine. However, the molecular mechanisms by which TGF-β exerts its effects on Th17 cell differentiation remain elusive. We report that TGF-β does not directly promote Th17 cell differentiation but instead acts indirectly by blocking expression of the transcription factors signal transducer and activator of transcription (STAT) 4 and GATA-3, thus preventing Th1 and Th2 cell differentiation. In contrast, TGF-β had no effect on the expression of retinoic acid receptor–related orphan nuclear receptor t, a Th17-specific transcription factor. Interestingly, in Stat-6^–/–T-bet^–/– mice, which are unable to generate Th1 and Th2 cells, IL-6 alone was sufficient to induce robust differentiation of Th17 cells, whereas TGF-β had no effect, suggesting that TGF-β is dispensable for Th17 cell development. Consequently, BALB/c Stat-6^–/–T-bet^–/– mice, but not wild-type BALB/c mice, were highly susceptible to the development of experimental autoimmune encephalomyelitis, which could be blocked by anti–IL-17 antibodies but not by anti–TGF-β antibodies. Collectively, these data provide evidence that TGF-β is not directly required for the molecular orchestration of Th17 cell differentiation.

Staphylococcus aureus synthesizes adenosine to escape host immune responses

Thammavongsa, V., Kern, J. W., Missiakas, D. M., Schneewind, O. — Mon, 26 Oct 2009 09:19:00 PDT

Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses.

Subtilase cytotoxin cleaves newly synthesized BiP and blocks antibody secretion in B lymphocytes

Mon, 26 Oct 2009 09:19:00 PDT

Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain–binding protein (BiP) to yield a C-terminal fragment that contains BiP’s substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus "aged" BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved.

Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes

Mon, 26 Oct 2009 09:19:00 PDT

In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)–mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study has characterized the role of PDK1 in the pathology caused by deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN). PDK1 is shown to be essential for lymphomagenesis caused by deletion of PTEN in T cell progenitors. However, PTEN deletion bypasses the normal PDK1-controlled signaling pathways that determine thymocyte growth and proliferation. PDK1 does have important functions in PTEN-null thymocytes, notably to control the PKB–Foxo signaling axis and to direct the repertoire of adhesion and chemokine receptors expressed by PTEN-null T cells. The results thus provide two novel insights concerning pathological signaling caused by PTEN loss in lymphocytes. First, PTEN deletion bypasses the normal PDK1-controlled metabolic checkpoints that determine cell growth and proliferation. Second, PDK1 determines the cohort of chemokine and adhesion receptors expressed by PTEN-null cells, thereby controlling their migratory capacity.

Mast cell-derived particles deliver peripheral signals to remote lymph nodes

Mon, 26 Oct 2009 09:19:00 PDT

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

T-bet-dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow

Mon, 26 Oct 2009 09:19:00 PDT

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P₅) transcript levels, and S1P₅-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P₁ also plays a role in NK cell egress from LNs. S1P₅ is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P₅ as a T-bet–induced gene that is required for NK cell egress from LNs and BM.

Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow

Mon, 26 Oct 2009 09:19:00 PDT

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFR⁺Sca-1⁺CD45^–TER119^–) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

CD1-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice

Mon, 26 Oct 2009 09:19:00 PDT

Group 1 CD1 (CD1a, CD1b, and CD1c)–restricted T cells recognize mycobacterial lipid antigens and are found at higher frequencies in Mycobacterium tuberculosis (Mtb)–infected individuals. However, their role and dynamics during infection remain unknown because of the lack of a suitable small animal model. We have generated human group 1 CD1 transgenic (hCD1Tg) mice that express all three human group 1 CD1 isoforms and support the development of group 1 CD1–restricted T cells with diverse T cell receptor usage. Both mycobacterial infection and immunization with Mtb lipids elicit group 1 CD1–restricted Mtb lipid–specific T cell responses in hCD1Tg mice. In contrast to CD1d-restricted NKT cells, which rapidly respond to initial stimulation but exhibit anergy upon reexposure, group 1 CD1–restricted T cells exhibit delayed primary responses and more rapid secondary responses, similar to conventional T cells. Collectively, our data demonstrate that group 1 CD1–restricted T cells participate in adaptive immune responses upon mycobacterial infection and could serve as targets for the development of novel Mtb vaccines.

Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone

Mon, 26 Oct 2009 09:19:01 PDT

Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective treatment of the disease might require rescuing β cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional β cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-β–dependent emergence of autoantigen-specific regulatory T cells and recovery of β cell function in the diabetic recipients.

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

Hsu, L.-Y., Tan, Y. X., Xiao, Z., Malissen, M., Weiss, A. — Mon, 26 Oct 2009 09:19:01 PDT

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.

Leukotriene E4-induced pulmonary inflammation is mediated by the P2Y12 receptor

Mon, 26 Oct 2009 09:19:01 PDT

Of the potent lipid inflammatory mediators comprising the cysteinyl leukotrienes (LTs; LTC₄, LTD₄, and LTE₄), only LTE₄ is stable and abundant in vivo. Although LTE₄ shows negligible activity at the type 1 and 2 receptors for cys-LTs (CysLT₁R and CysLT₂R), it is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in humans with asthma. We show that the adenosine diphosphate (ADP)–reactive purinergic (P2Y₁₂) receptor is required for LTE₄-mediated pulmonary inflammation. P2Y₁₂ receptor expression permits LTE₄ -induced activation of extracellular signal-regulated kinase in Chinese hamster ovary cells and permits chemokine and prostaglandin D₂ production by LAD2 cells, a human mast cell line. P2Y₁₂ receptor expression by LAD2 cells is required for competition between radiolabeled ADP and unlabeled LTE₄ but not for direct binding of LTE₄, suggesting that P2Y₁₂ complexes with another receptor to recognize LTE₄. Administration of LTE₄ to the airways of sensitized mice potentiates eosinophilia, goblet cell metaplasia, and expression of interleukin-13 in response to low-dose aerosolized allergen. These responses persist in mice lacking both CysLT₁R and CysLT₂R but not in mice lacking P2Y₁₂ receptors. The effects of LTE₄ on P2Y₁₂ in the airway were abrogated by platelet depletion. Thus, the P2Y₁₂ receptor is required for proinflammatory actions of the stable abundant mediator LTE₄ and is a novel potential therapeutic target for asthma.

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Mon, 26 Oct 2009 09:19:01 PDT

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between "full-length" and "deleted" forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1⁺ and C2⁺ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4⁺ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.

Native and aspirin-triggered lipoxins control innate immunity by inducing proteasomal degradation of TRAF6

Mon, 26 Oct 2009 09:19:01 PDT

Interplay of Oct4 with Sox2 and Sox17: a molecular switch from stem cell pluripotency to specifying a cardiac fate

Stefanovic, S., Abboud, N., Desilets, S., Nury, D., Cowan, C., Puceat, M. — Mon, 28 Sep 2009 10:03:24 PDT

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Wang, T., Lao, U., Edgar, B. A. — Mon, 28 Sep 2009 10:03:24 PDT

The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER

Mon, 28 Sep 2009 10:03:24 PDT

The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice

Mon, 28 Sep 2009 10:03:24 PDT

Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes

Simonsen, A., Tooze, S. A. — Mon, 28 Sep 2009 10:03:24 PDT

PPAR{gamma} tackles treacherous T cells

Leslie, M. — Mon, 28 Sep 2009 10:03:23 PDT

EBV takes a toll

Leslie, M. — Mon, 28 Sep 2009 10:03:23 PDT

Suspected asthma mutation leaves mice gasping

Leslie, M. — Mon, 28 Sep 2009 10:03:23 PDT

Clingy bacteria and Crohn's disease

Leslie, M. — Mon, 28 Sep 2009 10:03:23 PDT

"Killer" pathway not guilty

Leslie, M. — Mon, 28 Sep 2009 10:03:23 PDT

Mickie Bhatia: Embryonic stem cells come of age

Maxmen, A. — Mon, 28 Sep 2009 10:03:23 PDT

Regulation of pathogenesis and immunity in helminth infections

Maizels, R. M., Pearce, E. J., Artis, D., Yazdanbakhsh, M., Wynn, T. A. — Mon, 28 Sep 2009 10:03:23 PDT

Helminths are multicellular eukaryotic parasites that infect over one quarter of the world’s population. Through coevolution with the human immune system, these organisms have learned to exploit immunoregulatory pathways, resulting in asymptomatic tolerance of infections in many individuals. When infections and the resulting immune responses become dysregulated, however, acute and chronic pathologies often develop. A recent international meeting focused on how these parasites modulate host immunity and how control of parasitic and immunopathological disease might be achieved.

Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis

Mon, 28 Sep 2009 10:03:23 PDT

Atherosclerosis is an inflammatory vascular disease responsible for the first cause of mortality worldwide. Recent studies have clearly highlighted the critical role of the immunoinflammatory balance in the modulation of disease development and progression. However, the immunoregulatory pathways that control atherosclerosis remain largely unknown. We show that loss of suppressor of cytokine signaling (SOCS) 3 in T cells increases both interleukin (IL)-17 and IL-10 production, induces an antiinflammatory macrophage phenotype, and leads to unexpected IL-17–dependent reduction in lesion development and vascular inflammation. In vivo administration of IL-17 reduces endothelial vascular cell adhesion molecule–1 expression and vascular T cell infiltration, and significantly limits atherosclerotic lesion development. In contrast, overexpression of SOCS3 in T cells reduces IL-17 and accelerates atherosclerosis. We also show that in human lesions, increased levels of signal transducer and activator of transcription (STAT) 3 phosphorylation and IL-17 are associated with a stable plaque phenotype. These results identify novel SOCS3-controlled IL-17 regulatory pathways in atherosclerosis and may have important implications for the understanding of the increased susceptibility to vascular inflammation in patients with dominant-negative STAT3 mutations and defective Th17 cell differentiation.

The nuclear receptor PPAR{gamma} selectively inhibits Th17 differentiation in a T cell-intrinsic fashion and suppresses CNS autoimmunity

Mon, 28 Sep 2009 10:03:23 PDT

T helper cells secreting interleukin (IL)-17 (Th17 cells) play a crucial role in autoimmune diseases like multiple sclerosis (MS). Th17 differentiation, which is induced by a combination of transforming growth factor (TGF)-β/IL-6 or IL-21, requires expression of the transcription factor retinoic acid receptor–related orphan receptor t (RORt). We identify the nuclear receptor peroxisome proliferator–activated receptor (PPAR) as a key negative regulator of human and mouse Th17 differentiation. PPAR activation in CD4⁺ T cells selectively suppressed Th17 differentiation, but not differentiation into Th1, Th2, or regulatory T cells. Control of Th17 differentiation by PPAR involved inhibition of TGF-β/IL-6–induced expression of RORt in T cells. Pharmacologic activation of PPAR prevented removal of the silencing mediator for retinoid and thyroid hormone receptors corepressor from the RORt promoter in T cells, thus interfering with RORt transcription. Both T cell–specific PPAR knockout and endogenous ligand activation revealed the physiological role of PPAR for continuous T cell–intrinsic control of Th17 differentiation and development of autoimmunity. Importantly, human CD4⁺ T cells from healthy controls and MS patients were strongly susceptible to PPAR-mediated suppression of Th17 differentiation. In summary, we report a PPAR-mediated T cell–intrinsic molecular mechanism that selectively controls Th17 differentiation in mice and in humans and that is amenable to pharmacologic modulation. We therefore propose that PPAR represents a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS.

Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3

Mon, 28 Sep 2009 10:03:23 PDT

Epstein-Barr virus–encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)–like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induce signaling from TLR3, was released from EBV-infected cells, and the majority of the released EBER existed as a complex with a cellular EBER-binding protein La, suggesting that EBER was released from the cells by active secretion of La. Sera from patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), whose general symptoms are caused by proinflammatory cytokines contained EBER, and addition of RNA purified from the sera into culture medium induced signaling from TLR3 in EBV-transformed lymphocytes and peripheral mononuclear cells. Furthermore, DCs treated with EBER showed mature phenotype and antigen presentation capacity. These findings suggest that EBER, which is released from EBV-infected cells, is responsible for immune activation by EBV, inducing type I IFN and proinflammatory cytokines. EBER-induced activation of innate immunity would account for immunopathologic diseases caused by active EBV infection.

NOD2 regulates hematopoietic cell function during graft-versus-host disease

Mon, 28 Sep 2009 10:03:23 PDT

Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2^–/– hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.

TSLP and IL-7 use two different mechanisms to regulate human CD4+ T cell homeostasis

Lu, N., Wang, Y.-H., Wang, Y.-H., Arima, K., Hanabuchi, S., Liu, Y.-J. — Mon, 28 Sep 2009 10:03:23 PDT

Whether thymic stromal lymphopoietin (TSLP) directly induces potent human CD4⁺ T cell proliferation and Th2 differentiation is unknown. We report that resting and activated CD4⁺ T cells expressed high levels of IL-7 receptor a chain but very low levels of TSLP receptor (TSLPR) when compared with levels expressed in myeloid dendritic cells (mDCs). This was confirmed by immunohistology and flow cytometry analyses showing that only a subset of mDCs, with more activated phenotypes, expressed TSLPR in human tonsils in vivo. IL-7 induced strong STAT1, -3, and -5 activation and promoted the proliferation of naive CD4⁺ T cells in the presence of anti-CD3 and anti-CD28 monoclonal antibodies, whereas TSLP induced weak STAT5 activation, associated with marginally improved cell survival and proliferation, but failed to induce cell expansion and Th2 differentiation. The effect of TSLP on enhancing strong human T cell proliferation was observed only when sorted naive CD4⁺ T cells were cultured with mDCs at levels as low as 0.5%. TSLP could only induce naive CD4⁺ T cells to differentiate into Th2 cells in the presence of allogeneic mDCs. These results demonstrate that IL-7 and TSLP use different mechanisms to regulate human CD4⁺ T cell homeostasis.

TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes

Leung, M. W.L., Shen, S., Lafaille, J. J. — Mon, 28 Sep 2009 10:03:23 PDT

Numerous studies have highlighted the importance of high-affinity interactions between T cell receptors (TCRs) and their ligands in the selection of Foxp3⁺ regulatory T cells (T reg cells). To determine the role of the TCR in directing T cells into the Foxp3⁺ lineage, we generated transgenic (Tg) mice expressing TCRs from Foxp3⁺ cells. Initial analyses of the TCR Tg mice crossed with RAG-deficient mice showed that the percentage of Foxp3⁺ cells was very low. However, intrathymic injection and bone marrow chimera experiments showed a saturable increase of the Foxp3⁺ population when T reg TCR Tg cells were present in low numbers. Furthermore, when analyzing whole thymi of T reg TCR Tg RAG-deficient mice, we found significantly more Foxp3⁺ cells than in conventional T cell TCR Tg mice. Our results indicate that although the TCR has an instructive role in determining Foxp3 expression, selection of Foxp3⁺ individual clones in the thymus is limited by a very small niche.

Retinoic acid can enhance conversion of naive into regulatory T cells independently of secreted cytokines

Mon, 28 Sep 2009 10:03:23 PDT

It has been reported that retinoic acid (RA) enhances regulatory T (T reg) cell conversion by inhibiting the secretion of cytokines that interfere with conversion. This report shows that these conclusions provide a partial explanation at best. First, RA not only interfered with cytokine secretion but also with the ability of these cytokines to inhibit T reg cell conversion of naive T cells. Furthermore, RA enhanced conversion even in the absence of inhibitory cytokines. The latter effect depended on the RA receptor (RAR) but did not require Smad3, despite the fact that RA enhanced Smad3 expression. The RAR1 isoform was not essential for RA-dependent enhancement of transforming growth factor β–driven conversion, suggesting that conversion can also be mediated by RAR2. Interleukin (IL)-6 strongly reduced RAR expression levels such that a deficiency of the predominant RAR1 isoform leaves too little RAR2 for RA to inhibit the generation of Th17 cells in the presence of IL-6.

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions

Zhu, S.-N., Chen, M., Jongstra-Bilen, J., Cybulsky, M. I. — Mon, 28 Sep 2009 10:03:23 PDT

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2'-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6C^high monocyte recruitment in low density lipoprotein receptor–null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti–GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.

XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development

Mon, 28 Sep 2009 10:03:23 PDT

The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1^fl/fl CD19^+/cre conditional knockout (XBP1^CD19) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1^CD19 mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1^CD19 mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.

Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease

Mon, 28 Sep 2009 10:03:24 PDT

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of nuclear factor B activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL-1, dermal macrophage function, or expression of the toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF) 3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8–deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of up-regulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8–deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency.

Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Mon, 28 Sep 2009 10:03:24 PDT

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-fimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma

Mon, 28 Sep 2009 10:03:24 PDT

Polymorphisms in the interleukin-4 receptor chain (IL-4R) have been linked to asthma incidence and severity, but a causal relationship has remained uncertain. In particular, a glutamine to arginine substitution at position 576 (Q576R) of IL-4R has been associated with severe asthma, especially in African Americans. We show that mice carrying the Q576R polymorphism exhibited intense allergen-induced airway inflammation and remodeling. The Q576R polymorphism did not affect proximal signal transducer and activator of transcription (STAT) 6 activation, but synergized with STAT6 in a gene target– and tissue-specific manner to mediate heightened expression of a subset of IL-4– and IL-13–responsive genes involved in allergic inflammation. Our findings indicate that the Q576R polymorphism directly promotes asthma in carrier populations by selectively augmenting IL-4R–dependent signaling.

A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis

Mon, 28 Sep 2009 10:03:24 PDT

The C-type lectin dendritic cell–specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.

The angiopietin-1-Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice

Kugathasan, L., Ray, J. B., Deng, Y., Rezaei, E., Dumont, D. J., Stewart, D. J. — Mon, 28 Sep 2009 10:03:24 PDT

The role of the angiopoietin-1 (Ang1)–Tie2 pathway in the pathogenesis of pulmonary arterial hypertension (PAH) is controversial. Although Ang1 is well known to prevent endothelial activation and injury in systemic vascular beds, this pathway has been suggested to mediate pulmonary vascular remodeling in PAH. Therefore, we used transgenic models to determine the effect of increased or decreased Tie2 activity on the development of PAH. We now report modest spontaneous elevation in right ventricular systolic pressure in Tie2-deficient mice (Tie2^+/–) compared with wild-type (WT) littermate controls, which was exacerbated upon chronic exposure to the clinically relevant PAH triggers, serotonin (5-HT) or interleukin-6 (IL-6). Moreover, overexpression of Ang1 in transgenic mice had no deleterious effect on pulmonary hemodynamics and, if anything, blunted the response to 5-HT. Exposure to 5-HT or IL-6 also decreased lung Ang1 expression, further reducing Tie2 activity and inducing pulmonary apoptosis in the Tie2^+/– group only. Similarly, cultured pulmonary artery endothelial cells subjected to Tie2 silencing demonstrated increased susceptibility to apoptosis after 5-HT treatment. Finally, treatment of Tie2-deficient mice with Z-VAD, a pan-caspase inhibitor, prevented the pulmonary hypertensive response to 5-HT. Thus, these findings firmly establish that endothelial survival signaling via the Ang1–Tie2 pathway is protective in PAH.

Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection

Lee, S.-H., Kim, K.-S., Fodil-Cornu, N., Vidal, S. M., Biron, C. A. — Mon, 28 Sep 2009 10:03:24 PDT

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H's role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h^–/–, perforin 1 (Prf1)^–/–, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h^–/–Prf1^–/–), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.

Self-class I MHC molecules support survival of naive CD8 T cells, but depress their functional sensitivity through regulation of CD8 expression levels

Takada, K., Jameson, S. C. — Mon, 28 Sep 2009 10:03:24 PDT

Previous studies have suggested that naive CD8 T cells require self-peptide–major histocompatability complex (MHC) complexes for maintenance. However, interpretation of such studies is complicated because of the involvement of lymphopenic animals, as lymphopenia drastically alters naive T cell homeostasis and function. In this study, we explored naive CD8 T cell survival and function in nonlymphopenic conditions by using bone marrow chimeric donors and hosts in which class I MHC expression is absent or limited to radiosensitive versus radioresistant cells. We found that long-term survival of naive CD8 T cells (but not CD4 T cells) was impaired in the absence of class I MHC. However, distinct from this effect, class I MHC deprivation also enhanced naive CD8 T cell responsiveness to low-affinity (but not high-affinity) peptide–MHC ligands. We found that this improved sensitivity was a consequence of up-regulated CD8 levels, which was mediated through a transcriptional mechanism. Hence, our data suggest that, in a nonlymphopenic setting, self-class I MHC molecules support CD8 T cell survival, but that these interactions also attenuate naive T cell sensitivity by dynamic tuning of CD8 levels.

Distinct roles for E12 and E47 in B cell specification and the sequential rearrangement of immunoglobulin light chain loci

Beck, K., Peak, M. M., Ota, T., Nemazee, D., Murre, C. — Mon, 28 Sep 2009 10:03:24 PDT

The E2A gene products, E12 and E47, are critical regulators of B cell development. However, it remains elusive whether E12 and E47 have overlapping and/or distinct functions during B lymphopoiesis. We have generated mice deficient for either E12 or E47 and examined their roles in B cell maturation. We show that E47 is essential for developmental progression at the prepro–B cell stage, whereas E12 is dispensable for early B cell development, commitment, and maintenance. In contrast, both E12 and E47 play critical roles in pre–B and immature B cells to promote immunoglobulin (Ig) germline transcription as well as Ig VJ gene rearrangement. Furthermore, we show that E12 as well as E47 is required to promote receptor editing upon exposure to self-antigen. We demonstrate that increasing levels of E12 and E47 act to induce Ig germline transcription, promote trimethylated lysine 4 on histone 3 (H3) as well as H3 acetylation across the J region, and activate Ig VJ gene rearrangement. We propose that in the pre–B and immature B cell compartments, gradients of E12 and E47 activities are established to mechanistically regulate the sequential rearrangement of the Ig light chain genes.

Nonaminoglycoside compounds induce readthrough of nonsense mutations

Mon, 28 Sep 2009 10:03:24 PDT

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ~34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.

Blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade

Mon, 28 Sep 2009 10:03:24 PDT

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

Mon, 28 Sep 2009 10:03:24 PDT

Real-time in vivo imaging of p16Ink4a reveals cross talk with p53

Mon, 31 Aug 2009 10:07:14 PDT

The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins

Mon, 31 Aug 2009 10:07:14 PDT

Age-induced T cell troubles

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

Interferin' with shock

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

Giving gut DCs personality

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

DC-T reg cell checks and balances

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

Strep's key to the brain

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

Ajit Varki: On the origin of maladies

Maxmen, A. — Mon, 31 Aug 2009 10:07:13 PDT

Revisiting Crohn's disease as a primary immunodeficiency of macrophages

Casanova, J.-L., Abel, L. — Mon, 31 Aug 2009 10:07:13 PDT

Despite two decades of mouse immunology and human genetics studies, the pathogenesis of Crohn's disease (CD) remains elusive. New clinical investigations suggest that CD may be caused by inborn errors of macrophages. These errors may result in impaired attraction of granulocytes to the gut wall, causing impaired clearance of intruding bacteria, thereby precipitating the formation of granulomas. This theory paves the way for a macrophage-based Mendelian genetic dissection of CD.

The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion

Mon, 31 Aug 2009 10:07:13 PDT

In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood–brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN–BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen.

Feedback control of regulatory T cell homeostasis by dendritic cells in vivo

Mon, 31 Aug 2009 10:07:14 PDT

CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells (T reg cells) maintain self-tolerance and suppress autoimmune diseases such as type 1 diabetes and inflammatory bowel disease (IBD). In addition to their effects on T cells, T reg cells are essential for maintaining normal numbers of dendritic cells (DCs): when T reg cells are depleted, there is a compensatory Flt3-dependent increase in DCs. However, little is known about how T reg cell homeostasis is maintained in vivo. We demonstrate the existence of a feedback regulatory loop between DCs and T reg cells. We find that loss of DCs leads to a loss of T reg cells, and that the remaining T reg cells exhibit decreased Foxp3 expression. The DC-dependent loss in T reg cells leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon and interleukin 17. Conversely, increasing the number of DCs leads to increased T reg cell division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in T reg cells induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies.

TPL-2 negatively regulates interferon-{beta} production in macrophages and myeloid dendritic cells

Mon, 31 Aug 2009 10:07:14 PDT

Stimulation of Toll-like receptors (TLRs) on macrophages and dendritic cells (DCs) by pathogen-derived products induces the production of cytokines, which play an important role in immune responses. Here, we investigated the role of the TPL-2 signaling pathway in TLR induction of interferon-β (IFN-β) and interleukin-10 (IL-10) in these cell types. It has previously been suggested that IFN-β and IL-10 are coordinately regulated after TLR stimulation. However, in the absence of TPL-2 signaling, lipopolysaccharide (TLR4) and CpG (TLR9) stimulation resulted in increased production of IFN-β while decreasing IL-10 production by both macrophages and myeloid DCs. In contrast, CpG induction of both IFN- and IFN-β by plasmacytoid DCs was decreased in the absence of TPL-2, although extracellular signal-regulated kinase (ERK) activation was blocked. Extracellular signal-related kinase–dependent negative regulation of IFN-β in macrophages was IL-10–independent, required protein synthesis, and was recapitulated in TPL-2–deficient myeloid DCs by retroviral transduction of the ERK-dependent transcription factor c-fos.

Type I interferon drives tumor necrosis factor-induced lethal shock

Mon, 31 Aug 2009 10:07:14 PDT

Tumor necrosis factor (TNF) is reputed to have very powerful antitumor effects, but it is also a strong proinflammatory cytokine. Injection of TNF in humans and mice leads to a systemic inflammatory response syndrome with major effects on liver and bowels. TNF is also a central mediator in several inflammatory diseases. We report that type I interferons (IFNs) are essential mediators of the lethal response to TNF. Mice deficient in the IFN- receptor 1 (IFNAR-1) or in IFN-β are remarkably resistant to TNF-induced hypothermia and death. After TNF injection, IFNAR-1^–/– mice produced less IL-6, had less bowel damage, and had less apoptosis of enterocytes and hepatocytes compared with wild-type (WT) mice. Extensive gene expression analysis in livers of WT and IFNAR-1^–/– mice revealed a large deficiency in the response to TNF in the knockout mice, especially of IFN-stimulated response element–dependent genes, many of which encode chemokines. In livers of IFNAR-1^–/– mice, fewer infiltrating white blood cells (WBCs) were detected by immunohistochemistry. Deficiency of type I IFN signaling provided sufficient protection for potentially safer therapeutic use of TNF in tumor-bearing mice. Our data illustrate that type I IFNs act as essential mediators in TNF-induced lethal inflammatory shock, possibly by enhancing cell death and inducing chemokines and WBC infiltration in tissues.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

Mon, 31 Aug 2009 10:07:14 PDT

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of ¹¹¹In-labeled neutrophils at these sites and clearance of ³²P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

Mon, 31 Aug 2009 10:07:14 PDT

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor B, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

Mon, 31 Aug 2009 10:07:14 PDT

Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients.

Decreased TNF-{alpha} synthesis by macrophages restricts cutaneous immunosurveillance by memory CD4+ T cells during aging

Mon, 31 Aug 2009 10:07:14 PDT

Immunity declines during aging, however the mechanisms involved in this decline are not known. In this study, we show that cutaneous delayed type hypersensitivity (DTH) responses to recall antigens are significantly decreased in older individuals. However, this is not related to CC chemokine receptor 4, cutaneous lymphocyte-associated antigen, or CD11a expression by CD4⁺ T cells or their physical capacity for migration. Instead, there is defective activation of dermal blood vessels in older subject that results from decreased TNF- secretion by macrophages. This prevents memory T cell entry into the skin after antigen challenge. However, isolated cutaneous macrophages from these subjects can be induced to secrete TNF- after stimulation with Toll-like receptor (TLR) 1/2 or TLR 4 ligands in vitro, indicating that the defect is reversible. The decreased conditioning of tissue microenvironments by macrophage-derived cytokines may therefore lead to defective immunosurveillance by memory T cells. This may be a predisposing factor for the development of malignancy and infection in the skin during aging.

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy

Mon, 31 Aug 2009 10:07:14 PDT

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qβ-derived virus-like particles (Qβ–Fel d1). Qβ–Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcRIIb. In addition, treatment with Qβ–Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qβ–Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qβ–Fel d1 may be a safe and effective treatment for cat allergy.

Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration

Mon, 31 Aug 2009 10:07:14 PDT

FLT3-ITD–mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD–mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD–induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12–CXCR4 signaling. Through small interfering RNA–mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

Augmented TLR9-induced Btk activation in PIR-B-deficient B-1 cells provokes excessive autoantibody production and autoimmunity

Mon, 31 Aug 2009 10:07:14 PDT

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton's tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor B p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb^–/–) mice was further augmented in combination with the Fas^lpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.

Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Mon, 31 Aug 2009 10:07:14 PDT

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN- without inducing maturation or the production of IL-6 and TNF-. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF- and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis.

A role for CD47 in the development of experimental colitis mediated by SIRP{alpha}+CD103- dendritic cells

Mon, 31 Aug 2009 10:07:14 PDT

Mesenteric lymph node (mLN) CD103 (E integrin)⁺ dendritic cells (DCs) induce regulatory T cells and gut tolerance. However, the function of intestinal CD103^– DCs remains to be clarified. CD47 is the ligand of signal regulatory protein (SIRP) and promotes SIRP⁺ myeloid cell migration. We first show that mucosal CD103^– DCs selectively express SIRP and that their frequency was augmented in the lamina propria and mLNs of mice that developed Th17-biased colitis in response to trinitrobenzene sulfonic acid. In contrast, the percentage of SIRP⁺CD103^– DCs and Th17 responses were decreased in CD47-deficient (CD47 knockout [KO]) mice, which remained protected from colitis. We next demonstrate that transferring wild-type (WT), but not CD47 KO, SIRP⁺CD103^– DCs in CD47 KO mice elicited severe Th17-associated wasting disease. CD47 expression was required on the SIRP⁺CD103^– DCs for efficient trafficking to mLNs in vivo, whereas it was dispensable on both DCs and T cells for Th17 polarization in vitro. Finally, administration of a CD47-Fc molecule resulted in reduced SIRP⁺CD103^– DC–mediated Th17 responses and the protection of WT mice from colitis. We thus propose SIRP⁺CD103^– DCs as a pathogenic DC subset that drives Th17-biased responses and colitis, and the CD47–SIRP axis as a potential therapeutic target for inflammatory bowel disease.

Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals

Mon, 31 Aug 2009 10:07:14 PDT

The hypothesis that bystander inflammatory signals promote memory B cell (B_MEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220⁺IgG⁺ B_MEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent B_MEM clonally expand. Surprisingly, proliferating B_MEM do not acquire germinal center (GC) B cell markers before generating daughter B_MEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of B_MEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of B_MEM occurred. The absence of a B_MEM response to nonspecific inflammatory signals clearly shows that B_MEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

Aryl hydrocarbon receptor in combination with Stat1 regulates LPS-induced inflammatory responses

Mon, 31 Aug 2009 10:07:14 PDT

Toll-like receptor (TLR) signals perform a crucial role in innate immune responses to pathogens. In this study, we found that the aryl hydrocarbon receptor (Ahr) negatively regulates inflammatory responses mediated by lipopolysaccharide (LPS) in macrophages. Ahr was induced in macrophages stimulated by LPS, but not by transforming growth factor (TGF)-β plus interleukin (IL)-6, which can induce Ahr in naive T cells. The production of IL-6 and tumor necrosis factor (TNF)- by LPS was significantly elevated in Ahr-deficient macrophages compared with that in wild-type (WT) cells. Ahr-deficient mice were more highly sensitive to LPS-induced lethal shock than WT mice. Signal transducer and activator of transcription 1 (Stat1) deficiency, as well as Ahr deficiency, augmented LPS-induced IL-6 production. We found that Ahr forms a complex with Stat1 and nuclear factor-kappa B (NF-B) in macrophages stimulated by LPS, which leads to inhibition of the promoter activity of IL-6. Ahr thus plays an essential role in the negative regulation of the LPS signaling pathway through interaction with Stat1.

Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection

Mon, 31 Aug 2009 10:07:14 PDT

Innate immune cells detect pathogens via pattern recognition receptors (PRRs), which signal for initiation of immune responses to infection. Studies with Dectin-1, a PRR for fungi, have defined a novel innate signaling pathway involving Syk kinase and the adaptor CARD9, which is critical for inducing Th17 responses to fungal infection. We show that another C-type lectin, Dectin-2, also signals via Syk and CARD9, and contributes to dendritic cell (DC) activation by fungal particles. Unlike Dectin-1, Dectin-2 couples to Syk indirectly, through association with the FcR chain. In a model of Candida albicans infection, blockade of Dectin-2 did not affect innate immune resistance but abrogated Candida-specific T cell production of IL-17 and, in combination with the absence of Dectin-1, decreased Th1 responses to the organism. Thus, Dectin-2 constitutes a major fungal PRR that can couple to the Syk–CARD9 innate signaling pathway to activate DCs and regulate adaptive immune responses to fungal infection.

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Mon, 03 Aug 2009 10:02:15 PDT

Cohesin regulates VSG monoallelic expression in trypanosomes

Landeira, D., Bart, J.-M., Van Tyne, D., Navarro, M. — Mon, 03 Aug 2009 10:02:15 PDT

Proteins are the new carbs

Maxmen, A. — Mon, 03 Aug 2009 10:02:14 PDT

IL-9 gets around

Maxmen, A. — Mon, 03 Aug 2009 10:02:14 PDT

Activin eases asthma

Leslie, M. — Mon, 03 Aug 2009 10:02:14 PDT

Cracking the schistosome egg

Leslie, M. — Mon, 03 Aug 2009 10:02:14 PDT

Neutrophils gone wild

Leslie, M. — Mon, 03 Aug 2009 10:02:14 PDT

Jacques Banchereau: On a quest for cures

Maxmen, A. — Mon, 03 Aug 2009 10:02:14 PDT

Nine lives: plasticity among T helper cell subsets

Locksley, R. M. — Mon, 03 Aug 2009 10:02:14 PDT

The division of labor among two types of T helper (Th) subsets, first described over 20 yr ago, has been buffeted by the discovery of new subsets and new cytokines that can be coaxed out of T cells with increasing disregard for the subset of origin. Although Th17 cells and regulatory T (T reg) cells are widely accepted subsets, and others are being proposed, their plasticity is difficult to reconcile with the definitions of Th subsets as put forth in the initial description of Th1 and Th2 cells. A deeper molecular context will be required to reconcile the ever-increasing complexity of effector T cells.

Vicious circle: systemic autoreactivity in Ro52/TRIM21-deficient mice

Bolland, S., Garcia-Sastre, A. — Mon, 03 Aug 2009 10:02:14 PDT

Dysregulated innate responses, particularly excessive activation of interferon (IFN) pathways, have been implicated in the development of autoimmune pathologies. Autoreactivity frequently targets IFN-inducible genes such as the Ro autoantigens, which ubiquitinate and inhibit interferon regulatory factors (IRFs). A new study validates the role of these common autoantigens in preventing autoimmunity. The findings reveal that injury-induced systemic autoimmune disease is exacerbated in the absence of Ro52/Trim21 and is driven by the IL-23–Th17 pathway.

IL-9 as a mediator of Th17-driven inflammatory disease

Mon, 03 Aug 2009 10:02:14 PDT

We report that like other T cells cultured in the presence of transforming growth factor (TGF) β, Th17 cells also produce interleukin (IL) 9. Th17 cells generated in vitro with IL-6 and TGF-β as well as purified ex vivo Th17 cells both produced IL-9. To determine if IL-9 has functional consequences in Th17-mediated inflammatory disease, we evaluated the role of IL-9 in the development and progression of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The data show that IL-9 neutralization and IL-9 receptor deficiency attenuates disease, and this correlates with decreases in Th17 cells and IL-6–producing macrophages in the central nervous system, as well as mast cell numbers in the regional lymph nodes. Collectively, these data implicate IL-9 as a Th17-derived cytokine that can contribute to inflammatory disease.

Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway

Mon, 03 Aug 2009 10:02:14 PDT

Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52^–/–), which appear phenotypically normal if left unmanipulated. However, Ro52^–/– mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52^–/– mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23–Th17 pathway.

Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses

Mon, 03 Aug 2009 10:02:14 PDT

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1–depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1–mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.

The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1)

Mon, 03 Aug 2009 10:02:15 PDT

Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition mouse dendritic cells (DCs) to promote Th2 lymphocyte differentiation. Using an in vitro bystander polarization assay as the readout, we purified and identified the major Th2-inducing component from soluble egg extract (SEA) as the secreted T2 ribonuclease, omega-1. The Th2-promoting activity of omega-1 was found to be sensitive to ribonuclease inhibition and did not require MyD88/TRIF signaling in DCs. In common with unfractioned SEA, the purified native protein suppresses lipopolysaccharide-induced DC activation, but unlike SEA, it fails to trigger interleukin 4 production from basophils. Importantly, omega-1–exposed DCs displayed pronounced cytoskeletal changes and exhibited decreased antigen-dependent conjugate formation with CD4⁺ T cells. Based on this evidence, we hypothesize that S. mansoni omega-1 acts by limiting the interaction of DCs with CD4⁺ T lymphocytes, thereby lowering the strength of the activation signal delivered.

Group B Streptococcus suppression of phagocyte functions by protein-mediated engagement of human Siglec-5

Mon, 03 Aug 2009 10:02:15 PDT

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface. We recently demonstrated that GBS Sia can bind human CD33-related Sia-recognizing immunoglobulin (Ig) superfamily lectins (hCD33rSiglecs), a family of inhibitory receptors expressed on the surface of leukocytes. We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall–anchored β protein, resulting in recruitment of SHP protein tyrosine phosphatases. Using a panel of WT and mutant GBS strains together with Siglec-expressing cells and soluble Siglec-Fc chimeras, we show that GBS β protein binding to Siglec-5 functions to impair human leukocyte phagocytosis, oxidative burst, and extracellular trap production, promoting bacterial survival. We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia

Mon, 03 Aug 2009 10:02:15 PDT

We identify an autosomal mutation in the CSF3R gene in a family with a chronic neutrophilia. This T617N mutation energetically favors dimerization of the granulocyte colony-stimulating factor (G-CSF) receptor transmembrane domain, and thus, strongly promotes constitutive activation of the receptor and hypersensitivity to G-CSF for proliferation and differentiation, which ultimately leads to chronic neutrophilia. Mutant hematopoietic stem cells yield a myeloproliferative-like disorder in xenotransplantation and syngenic mouse bone marrow engraftment assays. The survey of 12 affected individuals during three generations indicates that only one patient had a myelodysplastic syndrome. Our data thus indicate that mutations in the CSF3R gene can be responsible for hereditary neutrophilia mimicking a myeloproliferative disorder.

Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide

Mon, 03 Aug 2009 10:02:15 PDT

Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor B, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies

Peggs, K. S., Quezada, S. A., Chambers, C. A., Korman, A. J., Allison, J. P. — Mon, 03 Aug 2009 10:02:15 PDT

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is a critical negative regulator of immune responses. Uniquely among known inhibitory receptors, its genetic ablation results in a fulminating and fatal lymphoproliferative disorder. This central regulatory role led to the development of antibodies designed to block CTLA-4 activity in vivo, aiming to enhance immune responses against cancer. Despite their preclinical efficacy and promising clinical activity against late stage metastatic melanoma, the critical cellular targets for their activity remains unclear. In particular, debate has focused on whether the effector T cell (T_eff) or regulatory T cell (T reg cell) compartment is the primary target of antibody-mediated blockade. We developed a mouse expressing human instead of mouse CTLA-4, allowing us to evaluate the independent contributions of CTLA-4 blockade of each T cell compartment during cancer immunotherapy in an in vivo model of mouse melanoma. The data show that although blockade on effector cells significantly improves tumor protection, unicompartmental blockade on regulatory cells completely fails to enhance antitumor responses. However, concomitant blockade of both compartments leads to a synergistic effect and maximal antitumor activity. We conclude that the combination of direct enhancement of T_eff cell function and concomitant inhibition of T reg cell activity through blockade of CTLA-4 on both cell types is essential for mediating the full therapeutic effects of anti–CTLA-4 antibodies during cancer immunotherapy.

Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage

Mon, 03 Aug 2009 10:02:15 PDT

Nuclear factor B (NF-B) is one of the main transcription factors involved in regulating apoptosis, inflammation, chronic liver disease, and cancer progression. The IKK complex mediates NF-B activation and deletion of its regulatory subunit NEMO in hepatocytes (NEMO^hepa) triggers chronic inflammation and spontaneous hepatocellular carcinoma development. We show that NEMO^hepa mice were resistant to Fas-mediated apoptosis but hypersensitive to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) as the result of a strong up-regulation of its receptor DR5 on hepatocytes. Additionally, natural killer (NK) cells, the main source of TRAIL, were activated in NEMO^hepa livers. Interestingly, depletion of the NK1.1⁺ cells promoted a significant reduction of liver inflammation and an improvement of liver histology in NEMO^hepa mice. Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)–mediated injury. The critical role of the NK cell/TRAIL axis in NEMO^hepa livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient^–/– mononuclear cells. Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling. As this mechanism is important in human liver diseases, NEMO^hepa mice are an interesting tool to give insight into liver pathophysiology and to develop future therapeutic strategies.

Pre-B cell receptor-mediated cell cycle arrest in Philadelphia chromosome-positive acute lymphoblastic leukemia requires IKAROS function

Mon, 03 Aug 2009 10:02:15 PDT

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph⁺) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph⁺ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph⁺ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph⁺ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression.

Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells

Mon, 03 Aug 2009 10:02:15 PDT

Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)–specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10–secreting CD4⁺ T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4⁺ T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)- to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN- and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

Activin-A induces regulatory T cells that suppress T helper cell immune responses and protect from allergic airway disease

Mon, 03 Aug 2009 10:02:15 PDT

Activin-A is a pleiotropic cytokine that participates in developmental, inflammatory, and tissue repair processes. Still, its effects on T helper (Th) cell–mediated immunity, critical for allergic and autoimmune diseases, are elusive. We provide evidence that endogenously produced activin-A suppresses antigen-specific Th2 responses and protects against airway hyperresponsiveness and allergic airway disease in mice. Importantly, we reveal that activin-A exerts suppressive function through induction of antigen-specific regulatory T cells that suppress Th2 responses in vitro and upon transfer in vivo. In fact, activin-A also suppresses Th1-driven responses, pointing to a broader immunoregulatory function. Blockade of interleukin 10 and transforming growth factor β1 reverses activin-A–induced suppression. Remarkably, transfer of activin-A–induced antigen-specific regulatory T cells confers protection against allergic airway disease. This beneficial effect is associated with dramatically decreased maturation of draining lymph node dendritic cells. Therapeutic administration of recombinant activin-A during pulmonary allergen challenge suppresses Th2 responses and protects from allergic disease. Finally, we demonstrate that immune cells infiltrating the lungs from individuals with active allergic asthma, and thus nonregulated inflammatory response, exhibit significantly decreased expression of activin-A's responsive elements. Our results uncover activin-A as a novel suppressive factor for Th immunity and a critical controller of allergic airway disease.

Myocyte necrosis underlies progressive myocardial dystrophy in mouse dsg2-related arrhythmogenic right ventricular cardiomyopathy

Mon, 03 Aug 2009 10:02:15 PDT

Mutations in the cardiac desmosomal protein desmoglein-2 (DSG2) are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). We studied the explanted heart of a proband carrying the DSG2-N266S mutation as well as transgenic mice (Tg-NS) with cardiac overexpression of the mouse equivalent of this mutation, N271S-dsg2, with the aim of investigating the pathophysiological mechanisms involved. Transgenic mice recapitulated the clinical features of ARVC, including sudden death at young age, spontaneous ventricular arrhythmias, cardiac dysfunction, and biventricular dilatation and aneurysms. Investigation of transgenic lines with different levels of transgene expression attested to a dose-dependent dominant-negative effect of the mutation. We demonstrate for the first time that myocyte necrosis is the key initiator of myocardial injury, triggering progressive myocardial damage, including an inflammatory response and massive calcification within the myocardium, followed by injury repair with fibrous tissue replacement, and myocardial atrophy. These observations were supported by findings in the explanted heart from the patient. Insight into mechanisms initiating myocardial damage in ARVC is a prerequisite to the future development of new therapies aimed at delaying onset or progression of the disease.

NF-{kappa}B activity marks cells engaged in receptor editing

Cadera, E. J., Wan, F., Amin, R. H., Nolla, H., Lenardo, M. J., Schlissel, M. S. — Mon, 03 Aug 2009 10:02:15 PDT

Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-B–dependent IB gene was replaced with a lacZ (β-gal) reporter complementary DNA (cDNA; IB^+/lacZ) suggests a potential role for NF-B in receptor editing. Sorted β-gal⁺ pre–B cells showed increased levels of various markers of receptor editing. In IB^+/lacZ reporter mice expressing either innocuous or self-specific knocked in BCRs, β-gal was preferentially expressed in pre–B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IB superrepressor in primary bone marrow cultures resulted in diminished germline and rearranged transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in β-gal⁺ pre–B cells. Because IRF4 is a target of NF-B and is required for receptor editing, we suggest that NF-B could be acting through IRF4 to regulate receptor editing.

S region sequence, RNA polymerase II, and histone modifications create chromatin accessibility during class switch recombination

Wang, L., Wuerffel, R., Feldman, S., Khamlichi, A. A., Kenter, A. L. — Mon, 03 Aug 2009 10:02:15 PDT

Immunoglobulin class switch recombination is governed by long-range interactions between enhancers and germline transcript promoters to activate transcription and modulate chromatin accessibility to activation-induced cytidine deaminase (AID). However, mechanisms leading to the differential targeting of AID to switch (S) regions but not to constant (C_H) regions remain unclear. We show that S and C_H regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively. Chromatin accessibility is superimposable with the activating histone modifications, which extend throughout S regions irrespective of length. High density elongating RNA polymerase II (RNAP II) is detected in S regions, suggesting that the transcription machinery has paused and stalling is abolished by deletion of the S region. We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility. Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

CD1c bypasses lysosomes to present a lipopeptide antigen with 12 amino acids

Mon, 03 Aug 2009 10:02:15 PDT