Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein–coupled receptors (S1PR1–S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2–/– mice had a rapid recovery from anaphylaxis. In contrast, Sphk1–/– mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1–/– mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2–/– mice from anaphylaxis, whereas S1pr2+/– mice showed partial recovery. Unlike Sphk2–/– mice, Sphk1–/– and S1pr2–/– mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.
Ana Olivera, Christoph Eisner, Yoshiaki Kitamura, Sandra Dillahunt, Laura Allende, Galina Tuymetova, Wendy Watford, Francoise Meylan, Susanne C. Diesner, Lingli Li, Jurgen Schnermann, Richard L. Proia, Juan Rivera
Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their β chain (β57); this results in the absence of a salt bridge between β57 and Argα76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argα76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3β. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 Å) electrostatic interactions existed among Argα76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue β57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.
Kenji Yoshida, Adam L. Corper, Rana Herro, Bana Jabri, Ian A. Wilson, Luc Teyton
Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular epithelial cell protein known to play a role in host defense at mucosal surfaces. Here we show that a ligand specific for NOD1, a peptide derived from peptidoglycan, initiates an unexpected signaling pathway in human epithelial cell lines that results in the production of type I IFN. Detailed analysis revealed the components of the signaling pathway. NOD1 binding to its ligand triggered activation of the serine-threonine kinase RICK, which was then able to bind TNF receptor–associated factor 3 (TRAF3). This in turn led to activation of TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) and the subsequent activation of IFN regulatory factor 7 (IRF7). IRF7 induced IFN-β production, which led to activation of a heterotrimeric transcription factor complex known as IFN-stimulated gene factor 3 (ISGF3) and the subsequent production of CXCL10 and additional type I IFN. In vivo studies showed that mice lacking the receptor for IFN-β or subjected to gene silencing of the ISGF3 component Stat1 exhibited decreased CXCL10 responses and increased susceptibility to Helicobacter pylori infection, phenotypes observed in NOD1-deficient mice. These studies thus establish that NOD1 can activate the ISGF3 signaling pathway that is usually associated with protection against viral infection to provide mice with robust type I IFN–mediated protection from H. pylori and possibly other mucosal infections.
Tomohiro Watanabe, Naoki Asano, Stefan Fichtner-Feigl, Peter L. Gorelick, Yoshihisa Tsuji, Yuko Matsumoto, Tsutomu Chiba, Ivan J. Fuss, Atsushi Kitani, Warren Strober
Bertrand Huard, Thomas McKee, Carine Bosshard, Stéphane Durual, Thomas Matthes, Samir Myit, Olivier Donze, Christophe Frossard, Carlo Chizzolini, Christiane Favre, Rudolf Zubler, Jean Philippe Guyot, Pascal Schneider, Eddy Roosnek
Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.
Chrystelle Couëdel, Christopher Roman, Alison Jones, Paolo Vezzoni, Anna Villa, Patricia Cortes
Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.
Menno C. van Zelm, Julie Smet, Brigitte Adams, Françoise Mascart, Liliane Schandené, Françoise Janssen, Alina Ferster, Chiung-Chi Kuo, Shoshana Levy, Jacques J.M. van Dongen, Mirjam van der Burg
Peptide loading of MHC class II (MHCII) molecules is directly catalyzed by the MHCII-like molecule HLA-DM (DM). Another MHCII-like molecule, HLA-DO (DO), associates with DM, thereby modulating DM function. The biological role of DO-mediated regulation of DM activity in vivo remains unknown; however, it has been postulated that DO expression dampens presentation of self antigens, thereby preventing inappropriate T cell activation that ultimately leads to autoimmunity. To test the idea that DO modulation of the MHCII self-peptide repertoire mediates self tolerance, we generated NOD mice that constitutively overexpressed DO in DCs (referred to herein as NOD.DO mice). NOD mice are a mouse model for type 1 diabetes, an autoimmune disease mediated by the destruction of insulin-secreting pancreatic β cells. Our studies showed that diabetes development was completely blocked in NOD.DO mice. Similar to NOD mice, NOD.DO animals selected a diabetogenic T cell repertoire, and the numbers and function of Tregs were normal. Indeed, immune system function in NOD.DO mice was equivalent to that in NOD mice. NOD.DO DCs, however, presented an altered MHCII-bound self-peptide repertoire, thereby preventing the activation of diabetogenic T cells and subsequent diabetes development. These studies show that DO expression can shape the overall MHCII self-peptide repertoire to promote T cell tolerance.
Woelsung Yi, Nilufer P. Seth, Tom Martillotti, Kai W. Wucherpfennig, Derek B. Sant’Angelo, Lisa K. Denzin
The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry–based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.
Jens Eberlein, Tom T. Nguyen, Francisco Victorino, Lucy Golden-Mason, Hugo R. Rosen, Dirk Homann
TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Rα, which contributed to high avidity, and decreased expression of programmed death–ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses.
Qing Zhu, Colt Egelston, Susan Gagnon, Yongjun Sui, Igor M. Belyakov, Dennis M. Klinman, Jay A. Berzofsky
TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.
Zubin M. Bamboat, Lee M. Ocuin, Vinod P. Balachandran, Hebroon Obaid, George Plitas, Ronald P. DeMatteo