Journal of Experimental Medicine Brief Definitive Reports

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.

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.

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.

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.