Glucagon-like peptide-1 (GLP-1) and the regulation of human invariant natural killer T cells: lessons from obesity, diabetes and psoriasis

Abstract

Aims/hypothesis: The innate immune cells, invariant natural killer T cells (iNKT cells), are implicated in the pathogenesis of psoriasis, an inflammatory condition associated with obesity and other metabolic diseases, such as diabetes and dyslipidaemia. We observed an improvement in psoriasis severity in a patient within days of starting treatment with an incretin-mimetic, glucagon-like peptide-1 (GLP-1) receptor agonist. This was independent of change in glycaemic control. We proposed that this unexpected clinical outcome resulted from a direct effect of GLP-1 on iNKT cells.

Methods: We measured circulating and psoriatic plaque iNKT cell numbers in two patients with type 2 diabetes and psoriasis before and after commencing GLP-1 analogue therapy. In addition, we investigated the in vitro effects of GLP-1 on iNKT cells and looked for a functional GLP-1 receptor on these cells.

Results: The Psoriasis Area and Severity Index improved in both patients following 6 weeks of GLP-1 analogue therapy. This was associated with an alteration in iNKT cell number, with an increased number in the circulation and a decreased number in psoriatic plaques. The GLP-1 receptor was expressed on iNKT cells, and GLP-1 induced a dose-dependent inhibition of iNKT cell cytokine secretion, but not cytolytic degranulation in vitro.

Conclusions/interpretation: The clinical effect observed and the direct interaction between GLP-1 and the immune system raise the possibility of therapeutic applications for GLP-1 in inflammatory conditions such as psoriasis.

Fig. 1

iNKT cells express GLP-1R. a Expression of GLP1R mRNA by iNKT cell lines as determined by RT-PCR. Values were normalised to levels of β-actin (n = 4). b Quantification of expression of GLP1R by iNKT cells measured by real-time PCR; expression was also measured on a GLP-1R-negative HEK-293 cell line as a negative control; n = 3; ^† p < 0.0001. c Flow cytometry dot plots showing representative HEK-293 cell line, CD3⁺ T cell line and iNKT cell line stained with the mAb GLP-1R phycoerythrin (PE) and mAbs for either CD3 APC (T cells) or iNKT cell TCR FITC (iNKT cells). FMO, Flow Minus One control for each cell line. Gates were determined by FMO and unstained samples (not shown) (n = 5)

Fig. 2

Activation of GLP-1R on iNKT cells results in upregulation of cAMP and CREB phosphorylation. a Quantification of cAMP. iNKT cells (1.5 × 10⁶) were seeded in the presence or absence of plate-bound anti-CD3 (2 μg/ml) and cultured with the GLP-1 analogue (15 μg/ml) for the indicated times. As a positive control, cells were incubated for 15 min with the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (500 mol/l), and then stimulated for another 30 min with forskolin (30 μmol/l) cAMP. Cell extracts were prepared and assayed for levels of cAMP. ^† p < 0.0001. b iNKT cells (2 × 10⁶) were seeded in the presence or absence of plate-bound anti-CD3 (2 μg/ml) and cultured with the GLP-1 analogue (15 μg/ml) or lipopolysaccharide (LPS, 100 ng/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and analysed by western blotting using anti-phospho-CREB, anti-CREB and anti-β-actin antibodies

Fig. 3

GLP-1 modulates iNKT cell cytokines. IFN-γ (a) and IL-4 (b) production by iNKT cells (1 × 10⁵) after 24 h co-culture at a 1:1 ratio with CD1d-transfected C1R cells unloaded in the absence or presence of GLP-1 (150 μg/ml) (n = 5). IFN-γ (c) and IL-4 (d) production by iNKT cells after 24 h co-culture with CD1d-transfected C1R cells loaded or unloaded with 100 ng/ml of αGalCer in presence of increasing concentrations of GLP-1 (0–150 μg/ml, n = 3). Cytokine concentrations were measured by ELISA and analysed using GraphPad Prism (n = 3). *p < 0.05, ^† p < 0.005, ^‡ p < 0.0001

Fig. 4

GLP-1 modulation of iNKT cell cytokine production is GLP-1R-dependent. IFN-γ (a) and IL-4 (b) production by iNKT cells (1 × 10⁵) that were incubated for 1 h with or without 10 μg/ml exendin 9-39. Cells were then co-cultured at a 1:1 ratio with CD1d-transfected C1R cells unloaded or loaded with 100 ng/ml αGalCer in the presence of 150 μg/ml native GLP-1 for 24 h (n = 3). IFN-γ (c) and IL-4 (d) production by iNKT cells (1 × 10⁵) stimulated with PMA and ionomycin (Iono) in the absence or presence of 150 μg/ml native GLP-1 for 24 h (n = 3). Cytokine concentrations were measured by ELISA and analysed using GraphPad Prism. *p < 0.05, ^† p < 0.005, ^‡ p < 0.0001

Fig. 5

The GLP-1 analogue liraglutide modulates iNKT cell cytokine production, but not cytolytic degranulation of iNKT cells. a IFN-γ and (b) IL-4 production by iNKT cells (1 × 10⁵) co-cultured at a 1:1 ratio with CD1d-transfected C1R cells unloaded or loaded with 100 ng/ml of αGalCer in the presence of 15 μg/ml of the GLP-1 analogue. Cytokine concentrations were measured by ELISA and analysed using GraphPad Prism (n = 3). c Percentage of CD107-positive iNKT cells after culture with CD1d-transfected target cells using the HeLa cell line and (d) Jurkat cell line in the absence or presence of α-GalCer and GLP-1 analogue treatment. Results are means ± SEM of experiments using three different iNKT cell lines. ^† p < 0.0001