The level of FoxO1 and IL-15 in skeletal muscle, serum and synovial fluid in people with knee osteoarthritis: a case control study

Objectives Impaired muscle function is common in knee osteoarthritis (OA). Numerous biochemical molecules have been implicated in the development of OA; however, these have only been identified in the joint and serum. This study compared the expression of interleukin (IL-15) and Forkhead box protein-O1 (FoxO1) in muscle of patients with knee OA asymptomatic individuals, and examined whether IL-15 was also present in the joint and serum. Method Muscle and blood samples were collected from 19 patients with diagnosed knee OA and 10 age-matched asymptomatic individuals. Synovial fluid and muscle biopsies were collected from the OA group during knee replacement surgery. IL-15 and FoxO1were measured in the skeletal muscle. IL-15 abundance was also analysed in the serum of both groups and synovial fluid from the OA group. Knee extensor strength was measured and correlated with IL-15 and FoxO1 in the muscle. Results FoxO1 protein expression was higher (p=0.04), whereas IL-15 expression was lower (p=0.02) in the muscle of the OA group. Strength was also lower in the OA group, and was inversely correlated with FoxO1 expression. No correlation was found between IL-15 in the joint, muscle or serum. Conclusion Skeletal muscle, particularly the quadriceps, is affected in people with knee OA where elevated FoxO1 protein expression was associated with reduced muscle strength. While IL-15 protein expression in the muscle was lower in the knee OA group, no correlation was found between the expression of IL-15 protein in the muscle, joint and serum, which suggests that inflammation is regulated differently within these tissues.

Efeito da IL-15 sobre a atividade fungicida de neutrófilos humanos contra Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimierte biologische Aktivität allo- und tumorreaktiver CD8-+-T-Lymphozyten im humanisierten Mausmodell durch IL-15-Superagonisten

Interleukin 15 (IL-15) gilt als eines der vielversprechendsten zukünftigen Medikamente für die Krebstherapie. Es fördert die Proliferation, Persistenz und Funktion von CD8+ T-Zellen und vermittelt zahlreiche Effekte, die es als überlegene Alternative für das derzeit in der Klinik verwendete IL-2 erscheinen lassen. Für den Einsatz von IL-15 in der vorliegenden Arbeit wurde zunächst ein Protokoll zur Herstellung von rekombinantem IL-15 in E. coli etabliert. Das hergestellte Protein hatte eine zu kommerziellen Produkten vergleichbare Bioaktivität und begünstigte die Persistenz und Aktivität antigenspezifischer, humaner CD8+ T Zellen nach adoptivem Transfer in NSG-Mäuse, wobei unter anderem ein verstärkter Effekt auf T Zellen mit TSCM-Phänotyp beobachtet wurde. Um die Bioaktivität von IL-15 zu steigern, wurden super-agonistische IL-15-Fusions¬proteine entworfen und im Expi293-System hergestellt. Dabei wurde IL 15 kovalent mit der Sushi-Domäne, der IL-15Rα-Kette und einer IgG1-Fc-Domäne verbunden, was zu einer gesteigerten Affinität der IL 15-Superagonisten zum physiologischen, niederaffinen IL 15Rβγ und zu einer stark erhöhten Halbwertszeit in Mausserum führte. Die gesteigerte Affinität der IL-15-Super¬agonisten wurde durch die IL 15Rα-Sushi-Domäne vermittelt. Eine um 13 Amino¬säuren verlängerte Sushi-Domäne zeigte im Vergleich zur normalen Form eine nochmals ge¬steigerte Affinität. Die längere Halbwertszeit wurde von der Sushi- und der IgG1-Fc-Domäne vermittelt. Die IgG1-Fc-Domäne verstärkte die Wirkung der Fusionsproteine zusätzlich über einen Mechanismus, der wahrscheinlich mit der Transpräsentation durch Fc Re¬ze¬ptoren zusammen–hängt. Die gesteigerte Bioaktivität der IL-15-Superagonisten wurde im Tiermodell mit humanen und murinen T-Zellen bestätigt und ILR13+-Fc wurde als das Fusionsprotein mit der höchsten Bioaktivität identifiziert. Im Vergleich zu anderen IL-15-Superagonisten vereint es alle derzeit bekannten Eigenschaften zur Bioaktivitätssteigerung in einem einzigen Protein. In therapeutischen Versuchen mit adoptivem Transfer tumorreaktiver T-Zellen konnte der Antitumoreffekt durch ILR13+-Fc maßgeblich verstärkt werden. Als Modellsysteme wurden NSG-Mäuse, die mit humanen AML-Blasten oder einem soliden Ovarialkarzinom engraftet wurden, verwendet. Dabei wurden sowohl antigenspezifische als auch unspezifische Effekte beobachtet. Die unspezifischen Effekte wurden wahrscheinlich durch eine ILR13+-Fc-vermittelte Überexpression von NKG2D, einem Rezeptor der angeborenen Immunantwort, auf den adoptiv transferierten T Zellen vermittelt. Die Ergebnisse dieser Arbeit zeigen, dass IL-15 und die IL-15-Superagonisten die Proliferation und Reaktivität von CD8+ T-Zellen im Rahmen der Immuntherapie fördern können. Aufgrund der hohen Bioaktivität und potenzierten Wirksamkeit, könnten vor allem die IL 15-Superagonisten in Zukunft bei der Entwicklung effizienter Therapiemethoden eingesetzt werden und dadurch einen wichtigen Beitrag zu Behandlung von Krebs leisten. rnrn

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.

Defining the role of IL-15 trans-presentation by distinct cell-types during the development and homeostasis of Natural Killer and invariant Natural Killer T cells

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK cells, I revealed the non-hematopoietic compartment provides IL-15 to NK cells in the early stages of development while hematopoietic cells were crucial for the generation and maintenance of mature NK cells. Regarding iNKT cells in the thymus, IL-15 trans-presentation by non-hematopoietic cells was crucial for the survival of mature iNKT cells. In the liver, both hematopoietic and non-hematopoietic compartments provided IL-15 to both immature and mature iNKT cells. This IL-15 signal helped mediate the survival and proliferation of both NK and iNKT cells as well as induce the functional maturation of mature iNKT cells via enhanced T-bet expression. In conclusion, my work illustrates an important notion that the immunological niche of NK and iNKT cells is tightly regulated and that this regulation is meticulously influenced by the tissue microenvironment.

Determining the roles of dendritic cells and ICAM-1 in the transpresentation of IL-15 to CD8 T cells

The maintenance and generation of memory CD8 T cells is dependent on the cytokine IL-15. IL-15 is delivered by a novel mechanism termed transpresentation: IL-15 is presented by a cell expressing IL-15Ralpha to the CD8 T cell which responds via IL-2Rbeta/gammac. The identity of what cells transpresent IL-15 to support the survival and homeostatic proliferation of memory CD8 T cells is unknown. Using a transgenic mouse model that limits IL-15 transpresentation to DCs, I have demonstrated that DCs transpresent IL-15 to CD8 T cells. DCs transpresent IL-15 to CD8 T cells during the contraction of an immune response and also drive homeostatic proliferation of memory CD8 T cells. Additionally, I identified a role for ICAM-1 in promoting homeostatic proliferation. Wt memory CD8 T cells displayed impaired homeostatic proliferation in ICAM-1-/- hosts but not in models of acute IL-15-driven proliferation. In this way, the role of ICAM-1 in IL-15 transpresentation resembles the role for ICAM-1 in antigenpresentation: where antigen or IL-15 is limited, adhesion molecules are important for generating maximal responses. In vitro cultures between CD8 T cells and bone marrowdifferentiated DCs (BMDC) activated with a TLR agonist established a model of proliferation and signaling in CD8 T cells that was dependent on IL-15 transpresentation and required ICAM-1 expression by BMDCs. Regarding the expression of IL-15, I demonstrated that in normal mice it is undetectable without stimulation but is elevated in lymphopenic mice, suggesting a role for T cells in regulating IL-15 expression. Overall, these studies have identified many novel aspects of the interaction between DCs and CD8 T cells that were previously unknown. The study of adhesion molecules in IL-15 transpresentation describes a novel role for these well-known adhesion molecules and it will be interesting for future studies to further characterize this relationship for other IL-15-dependent cell types.

Determining the roles of dendritic cells and ICAM-1 in the transpresentation of IL-15 to CD8 T cells

The maintenance and generation of memory CD8 T cells is dependent on the cytokine IL-15. IL-15 is delivered by a novel mechanism termed transpresentation: IL-15 is presented by a cell expressing IL-15Ralpha to the CD8 T cell which responds via IL-2Rbeta/gammac. The identity of what cells transpresent IL-15 to support the survival and homeostatic proliferation of memory CD8 T cells is unknown. Using a transgenic mouse model that limits IL-15 transpresentation to DCs, I have demonstrated that DCs transpresent IL-15 to CD8 T cells. DCs transpresent IL-15 to CD8 T cells during the contraction of an immune response and also drive homeostatic proliferation of memory CD8 T cells. Additionally, I identified a role for ICAM-1 in promoting homeostatic proliferation. Wt memory CD8 T cells displayed impaired homeostatic proliferation in ICAM-1-/- hosts but not in models of acute IL-15-driven proliferation. In this way, the role of ICAM-1 in IL-15 transpresentation resembles the role for ICAM-1 in antigenpresentation: where antigen or IL-15 is limited, adhesion molecules are important for generating maximal responses. In vitro cultures between CD8 T cells and bone marrowdifferentiated DCs (BMDC) activated with a TLR agonist established a model of proliferation and signaling in CD8 T cells that was dependent on IL-15 transpresentation and required ICAM-1 expression by BMDCs. Regarding the expression of IL-15, I demonstrated that in normal mice it is undetectable without stimulation but is elevated in lymphopenic mice, suggesting a role for T cells in regulating IL-15 expression. Overall, these studies have identified many novel aspects of the interaction between DCs and CD8 T cells that were previously unknown. The study of adhesion molecules in IL-15 transpresentation describes a novel role for these well-known adhesion molecules and it will be interesting for future studies to further characterize this relationship for other IL-15-dependent cell types.

Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study

Assembly and mutual proximities of α, β, and γc subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2Rα, IL-2Rβ, and γc subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2–10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2Rβ-α, γ-α, and γ-β pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a “triangular model” in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific α receptors with the β and/or γc subunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice

IL-2 and -15 belong to the four α-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 105 plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-γ as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway

Rearrangements of the high mobility group protein I-C (HMGI-C) gene, consisting in the loss of the carboxyl-terminal tail, have been frequently detected in benign human tumors of mesenchymal origin. We have previously demonstrated that transgenic (TG) mice carrying a truncated HMGI-C construct (HMGI-C/T) exhibit a giant phenotype together with a predominantly abdominal/pelvic lipomatosis. Here, we report that HMGI-C/T TG mice develop natural killer (NK)-T/NK cell lymphomas starting from 12 months of age. We found an increased expression of IL-2 and IL-15 proteins and their receptors in these lymphomas, and we demonstrate that HMGI-C/T protein positively regulates their expression in vitro. Therefore, the HMGI-C/T-mediated chronic stimulation of the IL-2/IL-15 pathway could be responsible for the onset of NK-T/NK cell lymphomas in HMGI-C/T TG mice.

Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.

Étude de la signalisation virale de l'induction du gène de l'IL-15 dans les cellules monocytaires THP-1

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.