CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model.

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.

Clinical and Histopathological Amelioration of Experimental Autoimmune Encephalomyelitis by AAV Vectors Expressing a Soluble Interleukin-23 Receptor.

The role of the T helper (Th)17 pathway has been clearly demonstrated in the onset and progression of autoimmune diseases, where interleukin (IL)-23 is a key molecule in maintaining the response mediated by Th17 cells. As a consequence, recent strategies based on blocking the interaction between IL-23 and its receptor (IL-23R), for example the anti-p19 antibody tildrakizumab, have been developed to regulate the Th17 pathway from the initial stages of the disease. Here, a soluble (s)IL-23R cDNA was cloned in expression plasmids and viral vectors. The clinical efficacy of sIL-23R was evaluated in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis mice intravenously injected with a single dose of adeno-associated virus AAV8-sIL-23R vectors. Cytokine secretion was determined by multiplex assay, while histopathological analysis of the central nervous system was performed to study demyelination, inflammatory infiltration, and microglia and astroglia activation. We observed that administration of adeno-associated vector 8 encoding sIL-23R was associated with a significant disease improvement, including delay in the onset of the clinical signs; slower progress of the disease; interference with IL-23-mediated signal transducer and activator of transcription response by inhibiting of signal transducer and activator of transcription 3 phosphorylation; reduced demyelination and infiltration in the central nervous system; and lower astrocyte and microglia activation. Our results suggest that the use of vectors carrying sIL-23R to block the IL-23/IL-23R interaction may be a new therapeutic strategy for the treatment of multiple sclerosis.

IL-17A concentration of seminal plasma and follicular fluid in infertile men and women with various clinical diagnoses.

Seminal plasma and follicular fluid (FF) cytokine analysis are valuable tools for diagnoses and validation of therapeutic approaches for improving the chance of conception. Despite the initial discovery over a decade ago, the IL-17 family has not received much attention in the case of infertility. In this study, we analyzed the level of IL-17A in seminal plasma, follicular fluid and blood serum of infertile patients with different clinical diagnoses by Enzyme Linked Immunosorbent Assay (ELISA). The results showed that the level of IL-17A was higher in seminal plasma and blood serum of varicocele patients than the control group. The level of this cytokine was higher in follicular fluid of endometriosis, polycystic ovary syndrome (PCOS) and tubal factor patients than the control group. A similar elevation in IL-17A level was observed in blood serum of these patients. Furthermore, there was a correlation between the numbers of meiosis I (MI) oocytes and the level of blood serum and follicular fluid IL-17A in PCOS patients. Our data suggest a putative role of IL-17A in mediating these conditions and may have possible applications in the development of more effective diagnostic tools and therapeutic treatments for human reproductive disorders.

Differential effect of CD69 targeting on bystander and antigen-specific T cell proliferation.

In spite of an initially proposed role as a costimulatory molecule for CD69, in vivo studies showed it as a regulator of immune responses and lymphocyte egress. We found constitutive CD69 expression by T cell subsets and pDC. We examined a possible effect of CD69 on T cell proliferation using transfer models and in vitro assays. In mice locally expressing or receiving antigen, anti-CD692.2 treatment did not affect the proliferation of antigen-specific transgenic T cells in ADLN, although we observed the presence of proliferated T cells in non-ADLN and spleen. This was not affected by FTY720 treatment and thus, not contributed by increased egress of proliferated lymphocytes from ADLN. In the absence of antigen, anti-CD69 2.2 treatment induced bystander proliferation of transferred memory phenotype T cells. This proliferation was mediated by IL-2, as it was inhibited by anti-IL-2 or anti-CD25 antibodies in vitro and by anti-CD25 antibodies in vivo. It was also dependent on CD69 expression by donor T cells and recipient cells. CD69 targeting on T cells enhanced IL-2-mediated proliferation and CD25 expression. However, it did not lead to increased early IL-2 production by T cells. No T cell subset was found to be specifically required in the recipient. Instead, CD69 targeting on pDC induced their expression of IL-2 and CD25, and pDC depletion showed that this subset was involved in the proliferation induction. These results indicate that CD69 targeting induces bystander T cell proliferation through pDC IL-2 production and T cell sensitization to IL-2 without affecting antigen-driven T cell proliferation.

Structural and functional characterization of a novel nonglycosidic type I NKT agonist with immunomodulatory properties.

Activation of type I NKT (iNKT) cells by CD1d-presented agonists is a potent immunotherapeutic tool. α-Galactosylceramide (α-GalCer) is the prototypic agonist, but its excessive potency with simultaneous production of both pro- and anti-inflammatory cytokines hampers its potential therapeutic use. In search for novel agonists, we have analyzed the structure and function of HS44, a synthetic aminocyclitolic ceramide analog designed to avoid unrestrained iNKT cell activation. HS44 is a weaker agonist compared with α-GalCer in vitro, although in vivo it induces robust IFN-γ production, and highly reduced but still functional Th2 response. The characteristic cytokine storm produced upon α-GalCer activation was not induced. Consequently, HS44 induced a very efficient iNKT cell-dependent antitumoral response in B16 animal model. In addition, intranasal administration showed the capacity to induce lung inflammation and airway hyperreactivity, a cardinal asthma feature. Thus, HS44 is able to elicit functional Th1 or Th2 responses. Structural studies show that HS44 binds to CD1d with the same conformation as α-GalCer. The TCR binds to HS44 similarly as α-GalCer, but forms less contacts, thus explaining its weaker TCR affinity and, consequently, its weaker recognition by iNKT cells. The ability of this compound to activate an efficient, but not massive, tailored functional immune response makes it an attractive reagent for immune manipulation.

NKT TCR recognition of CD1d-α-C-galactosylceramide.

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vß8.2 and Vß7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vß8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.

Galacto-configured aminocyclitol phytoceramides are potent in vivo invariant natural killer T cell stimulators.

A new class of α-galactosylceramide (αGC) nonglycosidic analogues bearing galacto-configured aminocyclitols as sugar surrogates have been obtained. The aminocyclohexane having a hydroxyl substitution pattern similar to an α-galactoside is efficiently obtained by a sequence involving Evans aldol reaction and ring-closing metathesis with a Grubbs catalyst to give a key intermediate cyclohexene, which has been converted in galacto-aminocyclohexanes that are linked through a secondary amine to a phytoceramide lipid having a cerotyl N-acyl group. Natural Killer T (NKT) cellular assays have resulted in the identification of an active compound, HS161, which has been found to promote NKT cell expansion in vitro in a similar fashion but more weakly than αGC. This compound stimulates the release of Interferon-γ (IFNγ) and Interleukin-4 (IL-4) in iNKT cell culture but with lower potency than αGC. The activation of Invariant Natural Killer T (iNKT) cells by this compound has been confirmed in flow cytometry experiments. Remarkably, when tested in mice, HS161 selectively induces a very strong production of IFN-γ indicative of a potent Th1 cytokine profile. Overall, these data confirm the agonist activity of αGC lipid analogues having charged amino-substituted polar heads and their capacity to modulate the response arising from iNKT cell activation in vivo.