Circadian Clocks in Mouse and Human CD4+ T Cells

Though it has been shown that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression by qPCR in unstimulated CD4+ T cells and immune responses of PMA/ionomycin stimulated CD4+ T cells by FACS analysis purified from blood of healthy subjects at different time points throughout the day. Molecular clock as well as immune function was further analyzed in unstimulated T cells which were cultured in serum-free medium with circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, IL-2, IL-4, IFN-γ production and CD40L expression in freshly isolated CD4+ T cells. Further analysis of IFN-γ and CD40L in cultivated T cells revealed that these parameters remain rhythmic in vitro. Moreover, circadian luciferase reporter activity in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed regulation of the NF-κB pathway as a candidate mechanism mediating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic CD4+ T cell immune responses

Ligand-Induced Movements of Inner Transmembrane Helices of Glut1 Revealed by Chemical Cross-Linking of Di-Cysteine Mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site

Ganglioside GM3 Has an Essential Role in the Pathogenesis and Progression of Rheumatoid Arthritis

Rheumatoid arthritis (RA), a chronic systemic inflammatory disorder that principally attacks synovial joints, afflicts over 2 million people in the United States. Interleukin (IL)-17 is considered to be a master cytokine in chronic, destructive arthritis. Levels of the ganglioside GM3, one of the most primitive glycosphingolipids containing a sialic acid in the structure, are remarkably decreased in the synovium of patients with RA. Based on the increased cytokine secretions observed in in vitro experiments, GM3 might have an immunologic role. Here, to clarify the association between RA and GM3, we established a collagen-induced arthritis mouse model using the null mutation of the ganglioside GM3 synthase gene. GM3 deficiency exacerbated inflammatory arthritis in the mouse model of RA. In addition, disrupting GM3 induced T cell activation in vivo and promoted overproduction of the cytokines involved in RA. In contrast, the amount of the GM3 synthase gene transcript in the synovium was higher in patients with RA than in those with osteoarthritis. These findings indicate a crucial role for GM3 in the pathogenesis and progression of RA. Control of glycosphingolipids such as GM3 might therefore provide a novel therapeutic strategy for RA

AB0364 EFFICACY OF BARICITINIB (BARI) IN PATIENTS WITH RHEUMATOID ARTHRITIS (RA) WHOSE RESPONSE WAS INADEQUATE TO TOFACITINIB (TOFA).

Background:Currently, four types of JAK inhibitors are approved for the treatment of RA in Japan, however, they often show differences in clinical efficacies presumably due to their JAK selectivity.Objectives:To investigate the efficacy of Bari for patients with Tofa-inadequate response (IR), clinical profiles of seven Tofa IR patients were evaluated.Methods:We performed a single-center retrospective study on seven Tofa IR patients (female:7) who were switched to Bari. Items evaluated were as follows; patient’s baseline characteristics, continuation rate of Bari, swollen joint count, tender joint count, C-reactive protein (CRP), matrix proteinase 3 (MMP3), physician’s and patient’s visual analog scale (VAS), disease activity assessed by Disease Assessment Score of 28 joints - C-reactive protein (DAS 28-CRP), simplified disease activity index (SDAI) and clinical disease activity index (CDAI) at 2, 4, 8, 12 weeks, and the dosage of prednisone (PSL).Results:Patient’s mean age was 56.4 and mean disease duration was 9.2 years. Tofa had administered for 3.4 months (range 1-10) before switching to Bari. All patients were positive for both anti-citrullinated protein antibodies and rheumatoid factor. The number of prior biologics use was 2.3 (range1-4). 3 patients had concomitant MTX use (mean dosage 5.3 mg/week) and 4 had prednisone (mean dosage 3.3 mg/day). Mean swollen joint counts and tender joint counts were 5.0 (range 1-15) and 5.3 (range 1-15), respectively. Physician’s and Patient’s VAS 0-100 were 71 mm (range 50-90) and 73 mm (range 50-80), mean CRP levels were 1.6 mg/dl (range 0.01-4.3) and MMP3 levels were 357.3 mg/dl (range 27-933). Mean SDAI, CDAI and DAS-28-CRP were 26.3 (range17-50), 24.7 (range17-47) and 4.5, respectively. When enrolled, 4 of 7 patients corresponded to moderate disease activity (MDA) and 3 were in high disease activity (HDA) by all composite measures. After switched to Bari, the patient’s VAS significantly decreased at week 2 (P &lt;0.01). CDAI, SDAI and DAS28-CRP also showed a significant improvement at week 4, (P &lt;0.05). At week 4, 2 patients discontinued Bari due to the lack of efficacy or acute exacerbation of interstitial pneumonia. Thus, continuation rate of Bari at week 12 was 71.4%. Among 5 patients who continues Bari, all patients achieved low disease activity (LDA) by SDAI and CDAI at week 12. By DAS 28-CRP, 2 patients achieved MDA, 2 achieved LDA and 1 achieved remission (mean 2.6), respectively, at week 12, 3 of 4 patients taking PSL were able to withdraw that before week 12 (Figure).Conclusion:Result showed the efficacy of Bari for patients with Tofa IR, representing inadequate improvements of patient’s VAS with Tofa. The efficacy of Bari can be expected from the early stage and continued up to 12 weeks after switching. This difference in therapeutic effect may be due to each mechanism of action, Bari inhibits JAK1/2, whereas Tofa mainly inhibits JAK1 /3 (1). JAK2 is important for signal by GM-CSF, which has shown potential as an important therapeutic target in RA (2).Inhibition of JAK2 and GM-CSF may be the reason for Bari efficacy in Tofa IR patients.References:[1]O’Shea JJ et al. Back to the future: oral targeted therapy for RA and other autoimmune diseases. Nat Rev Rheumatol 2013 Mar;9(3):173-82.[2]Wicks IP et al. Targeting GM-CSF in inflammatory diseases. Nat Rev Rheumatol. 2016 Jan;12(1):37-48.Disclosure of Interests:None declared</jats:sec