Journal of Experimental Medicine Brief Definitive Reports

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

2008-05-12

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)–associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele–restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.

IL-6-dependent spontaneous proliferation is required for the induction of colitogenic IL-17-producing CD8+ T cells

2008-05-12

We propose a novel role for interleukin (IL) 6 in inducing rapid spontaneous proliferation (SP) of naive CD8⁺ T cells, which is a crucial step in the differentiation of colitogenic CD8⁺ T cells. Homeostasis of T cells is regulated by two distinct modes of cell proliferation: major histocompatibility complex/antigen–driven rapid SP and IL-7/IL-15–dependent slow homeostatic proliferation. Using our novel model of CD8⁺ T cell–dependent colitis, we found that SP of naive CD8⁺ T cells is essential for inducing pathogenic cytokine-producing effector T cells. The rapid SP was predominantly induced in mesenteric lymph nodes (LNs) but not in peripheral LNs under the influence of intestinal flora and IL-6. Indeed, this SP was markedly inhibited by treatment with anti–IL-6 receptor monoclonal antibody (IL-6R mAb) or antibiotic-induced flora depletion, but not by anti–IL-7R mAb and/or in IL-15–deficient conditions. Concomitantly with the inhibition of SP, anti–IL-6R mAb significantly inhibited the induction of CD8⁺ T cell–dependent autoimmune colitis. Notably, the transfer of naive CD8⁺ T cells derived from IL-17^–/– mice did not induce autoimmune colitis. Thus, we conclude that IL-6 signaling is crucial for SP under lymphopenic conditions, which subsequently caused severe IL-17–producing CD8⁺ T cell–mediated autoimmune colitis. We suggest that anti–IL-6R mAb may become a promising strategy for the therapy of colitis.

During negative selection, Nur77 family proteins translocate to mitochondria where they associate with Bcl-2 and expose its proapoptotic BH3 domain

Thompson, J., Winoto, A. — 2008-05-12

Apoptosis accompanying negative selection is a central but poorly understood event in T cell development. The Nur77 nuclear steroid receptor and Bim, a proapoptotic BH3-only member of the Bcl-2 family, are two molecules implicated in this process. However, how they relate to each other and how Nur77 induces apoptosis remain unclear. In thymocytes, Nur77 has been shown to induce cell death through a transcriptional-dependent pathway, but in cancer cell lines, Nur77 was reported to induce apoptosis through conversion of Bcl-2 into a killer protein at the mitochondria. Whether this Nur77 transcriptional-independent pathway actually occurs in vivo remains controversial. Using an optimized fractionation protocol for thymocytes, here we report that stimulation of CD4⁺CD8⁺ thymocytes results in translocation of Nur77 and its family member Nor-1 to the mitochondria, leading to their association with Bcl-2 and exposure of the Bcl-2 proapoptotic BH3 domain. In two T cell receptor transgenic models of negative selection, F5 and HY, a conformational change of the Bcl-2 molecule in the negatively selected T cell population was similarly observed. Thus, the Nur77 family and Bim pathways converge at mitochondria to mediate negative selection.

Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia

2008-04-14

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2

Okuno, T., Iizuka, Y., Okazaki, H., Yokomizo, T., Taguchi, R., Shimizu, T. — 2008-04-14

Activated blood platelets and macrophages metabolize prostaglandin H₂ into thromboxane A₂ and 12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid (12-HHT) in an equimolar ratio through the action of thromboxane synthase. Although it has been shown that 12-HHT is abundant in tissues and bodily fluids, this compound has long been viewed as a by-product lacking any specific function. We show that 12-HHT is a natural ligand for leukotriene B₄ (LTB₄) receptor-2 (BLT2), a G protein–coupled receptor that was originally identified as a low-affinity receptor for LTB₄. BLT2 agonistic activity in lipid fractions from rat small intestine was identified as 12-HHT using high-performance liquid chromatography and mass spectrometry. Exogenously expressed BLT2 in mammalian cells was activated by synthetic 12-HHT, as assessed by guanosine 5'-O-(3-thio) triphosphate binding, the activation of intracellular signaling pathways, and chemotaxis assay. Displacement analysis using [³H]LTB₄ showed that 12-HHT binds to BLT2 with a higher affinity than LTB₄. Lipid extracts from cyclooxygenase 1–deficient mice failed to activate BLT2. Bone marrow–derived mast cells (BMMCs) isolated from wild-type mice migrated toward a low concentration of 12-HHT, whereas BMMCs from BLT2-deficient mice did not. We conclude that 12-HHT is a natural lipid agonist of BLT2 in vivo and induces chemotaxis of mast cells.

Synthetic chemerin-derived peptides suppress inflammation through ChemR23

2008-04-14

Chemerin is a chemotactic protein that binds to the G protein–coupled receptor, ChemR23. We demonstrate that murine chemerin possesses potent antiinflammatory properties that are absolutely dependent on proteolytic processing. A series of peptides was designed, and only those identical to specific C-terminal chemerin sequences exerted antiinflammatory effects at picomolar concentrations in vitro. One of these, chemerin15 (C15; A¹⁴⁰-A¹⁵⁴), inhibited macrophage (M) activation to a similar extent as proteolyzed chemerin, but exhibited reduced activity as a M chemoattractant. Intraperitoneal administration of C15 (0.32 ng/kg) to mice before zymosan challenge conferred significant protection against zymosan-induced peritonitis, suppressing neutrophil (63%) and monocyte (62%) recruitment with a concomitant reduction in proinflammatory mediator expression. Importantly, C15 was unable to ameliorate zymosan-induced peritonitis in ChemR23^–/– mice, demonstrating that C15's antiinflammatory effects are entirely ChemR23 dependent. In addition, administration of neutralizing anti-chemerin antibody before zymosan challenge resulted in a significant exacerbation of peritoneal inflammation (up to 170%), suggesting an important endogenous antiinflammatory role for chemerin-derived species. Collectively, these results show that chemerin-derived peptides may represent a novel therapeutic strategy for the treatment of inflammatory diseases through ChemR23.

CXCR4 is required for the quiescence of primitive hematopoietic cells

Nie, Y., Han, Y.-C., Zou, Y.-R. — 2008-04-14

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4^–/– HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

A novel role for Lef-1, a central transcription mediator of Wnt signaling, in leukemogenesis

2008-03-17

Canonical Wnt signaling is critically involved in normal hematopoietic development and the self-renewal process of hematopoietic stem cells (HSCs). Deregulation of this pathway has been linked to a large variety of cancers, including different subtypes of leukemia. Lef-1 is a major transcription factor of this pathway and plays a pivotal role in lymphoid differentiation as well as in granulopoiesis. Here, we demonstrate Lef-1 expression in murine HSCs as well as its expression in human leukemia. Mice transplanted with bone marrow retrovirally transduced to express Lef-1 or a constitutive active Lef-1 mutant showed a severe disturbance of normal hematopoietic differentiation and finally developed B lymphoblastic and acute myeloid leukemia (AML). Lef-1–induced AMLs were characterized by immunoglobulin (Ig) DH-JH rearrangements and a promiscuous expression of lymphoid and myeloid regulatory factors. Furthermore, single cell experiments and limiting dilution transplantation assays demonstrated that Lef-1–induced AML was propagated by a leukemic stem cell with lymphoid characteristics displaying Ig DH-JH rearrangements and a B220⁺ myeloid marker^– immunophenotype. These data indicate a thus far unknown role of Lef-1 in the biology of acute leukemia, pointing to the necessity of balanced Lef-1 expression for an ordered hematopoietic development.

Intrathymic expression of Flt3 ligand enhances thymic recovery after irradiation

Kenins, L., Gill, J. W., Boyd, R. L., Hollander, G. A., Wodnar-Filipowicz, A. — 2008-03-17

Hematopoietic stem cell transplantation (HSCT) requires conditioning treatments such as irradiation, which leads to a severely delayed recovery of T cell immunity and constitutes a major complication of this therapy. Currently, our understanding of the mechanisms regulating thymic recovery is limited. It is known that a subpopulation of bone marrow (BM)–derived thymic immigrant cells and the earliest intrathymic progenitors express the FMS-like tyrosine kinase 3 (Flt3) receptor; however, the functional significance of this expression in the thymus is not known. We used the BM transplant model to investigate the importance of Flt3 ligand (FL) for the regeneration of the T cell compartment. We show that FL is expressed in the adult mouse thymus on the surface of perivascular fibroblasts. These cells surround the proposed thymic entry site of Flt3 receptor–positive T cell progenitors. After irradiation, perivascular FL expression is up-regulated and results in an enhanced recovery of thymic cellularity. Thymic grafting experiments confirm an intrathymic requirement for FL. Collectively, these results show that thymic stromal cell–mediated FL–Flt3 receptor interactions are important in the reconstitution of thymopoiesis early after lethal irradiation and HSCT, and provide a functional relevance to the expression of the Flt3 receptor on intrathymic T cell progenitors.

IL-10 blockade facilitates DNA vaccine-induced T cell responses and enhances clearance of persistent virus infection

Brooks, D. G., Lee, A. M., Elsaesser, H., McGavern, D. B., Oldstone, M. B.A. — 2008-03-17

Therapeutic vaccination is a potentially powerful strategy to establish immune control and eradicate persistent viral infections. Large and multifunctional antiviral T cell responses are associated with control of viral persistence; however, for reasons that were mostly unclear, current therapeutic vaccination approaches to restore T cell immunity and control viral infection have been ineffective. Herein, we confirmed that neutralization of the immunosuppressive factor interleukin (IL)-10 stimulated T cell responses and improved control of established persistent lymphocytic choriomeningitis virus (LCMV) infection. Importantly, blockade of IL-10 also allowed an otherwise ineffective therapeutic DNA vaccine to further stimulate antiviral immunity, thereby increasing T cell responses and enhancing clearance of persistent LCMV replication. We therefore propose that a reason that current therapeutic vaccination strategies fail to resurrect/sustain T cell responses is because they do not alleviate the immunosuppressive environment. Consequently, blocking key suppressive factors could render ineffective vaccines more efficient at improving T cell immunity, and thereby allow immune-mediated control of persistent viral infection.

RAGE signaling sustains inflammation and promotes tumor development

2008-02-18

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

Vaccine protection against Staphylococcus aureus pneumonia

Wardenburg, J. B., Schneewind, O. — 2008-02-18

Staphylococcus aureus pneumonia causes significant mortality in hospitalized or healthy individuals, and recent increases in morbidity are attributed to the rapid spread of methicillin-resistant S. aureus (MRSA) strains, which are often not susceptible to antibiotic therapy. -Hemolysin (Hla), a secreted pore-forming toxin, is an essential virulence factor of MRSA in a mouse model of S. aureus pneumonia. We show that the level of Hla expression by independent S. aureus strains directly correlates with their virulence. Active immunization with a mutant form of Hla (Hla_H35L), which cannot form pores, generates antigen-specific immunoglobulin G responses and affords protection against staphylococcal pneumonia. Moreover, transfer of Hla-specific antibodies protects naive animals against S. aureus challenge and prevents the injury of human lung epithelial cells during infection. Thus, Hla vaccination or immunotherapy may prevent S. aureus pneumonia in humans.

Periostin is essential for cardiac healingafter acute myocardial infarction

2008-02-18

Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin^–/– mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of smooth muscle actin–positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or v-integrin, which blocked the periostin-promoted cell migration, revealed that v-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI.

Forward genetic analysis of Toll-like receptor responses in wild-derived mice reveals a novel antiinflammatory role for IRAK1BP1

Conner, J. R., Smirnova, I. I., Poltorak, A. — 2008-02-18

Although inflammatory cytokines produced by activation of Toll-like receptors (TLRs) are essential for early host defense against infection, they also mediate a vast array of pathologies, including autoimmune disease, hypersensitivity reactions, and sepsis. Thus, numerous regulatory mechanisms exist in parallel with proinflammatory pathways to prevent excessive release of these potent effector molecules. We report elucidation of a novel regulatory function for interleukin receptor–associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1, also known as SIMPL) through quantitative trait locus mapping of the TLR response in wild-derived mouse strains. This gene emerged as a negative regulator of TLR2-mediated interleukin (IL)-6 production in MOLF/Ei mice, which expressed IRAK1BP1 mRNA in an allele-specific manner when crossed with the C57BL/6J strain. Human peripheral blood mononuclear cells and primary macrophages from two other wild-derived mouse strains also induced IRAK1BP1 mRNA by 4 hours after stimulation with agonists of various TLRs. Examination of its effects on IL-6 and other cytokines demonstrated that IRAK1BP1 regulates transcription of a specific subset of TLR-responsive genes, producing an overall antiinflammatory profile. Our results reveal that IRAK1BP1 is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. Furthermore, these results show that the genetic diversity of wild-derived mouse strains makes them a valuable model of important human gene functions that have been lost in some laboratory-inbred strains.

PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation

2008-02-18

Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor B activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.

Sustained desensitization to bacterial Toll-like receptor ligands after resolutionof respiratory influenza infection

2008-02-18

The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for ~35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-B activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.

Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases

2008-02-18

Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic–spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial–gut axis in human disease.

IL-33 reduces the development of atherosclerosis

2008-02-18

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1–like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE^–/– mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33–treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFN in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG₁, but decreased IgG_2a, which is consistent with a Th1-to-Th2 switch. IL-33–treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE^–/– mice compared with control IgG-treated mice. Furthermore, coadministration of an anti–IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.