Background: Clinical evidence connects hyperinsulinemia with obesity, poor lipid profile, and development of type 2 diabetes. However, its role in autoimmune conditions was questioned.

Objectives: In this study, we investigated effect of hyperinsulinemia on CD4 ⁺ T cell function in rheumatoid arthritis (RA) and the ability of the Jak-STAT inhibiting treatment (JAKi) to counteract this effect.

Methods: Plasma insulin levels were measured after an overnight lent in 56 female non-diabetic RA patients, by ELISA, and identified 12 patients with insulin levels above 157 pmol/L, indicating hyperinsulinemia ( Table 1 ). Transcriptional sequencing profile CD4+ cells and cytokine (IFNg, IL10, TNF, IL8, IL6) protein profile of CD4+ cells of patients with hyperinsulinemia using JAKi or no-JAKi treatment were compared. To investigate a direct effect of insulin and JAKi, on CD4+ cells proliferation by Cell-TraceViolet dilution and cell cycle and DNA enrichment by 7AAD staining of CD4+ cells from 14 healthy donors was studied by flow cytometry. Gene transcription was analyzed by qPCR and cytokine production by ELISA.

Results: CD4 ⁺ cells of patients with hyperinsulinemia had suppression of the PI3K-Src kinases and lower expression of Th subset specific transcription factors RORC, PRDM1 and STAT4, and cytokines IFNG, and IL10, suggesting a less aggressive CD4+ cell phenotype. Hyperinsulinemia patients had also lower serum levels of IL6 and lower erythrocyte sedimentation rate. Insulin exposure of CD4+ cells ex vivo decreased production of IFNγ, TNFα and IL6, along with decreased expression of IRS1 and PIK3CG, suggesting immunosuppressive abilities of insulin.

CD4 ⁺ cells of JAKi treated patients were recognized by increased insulin signaling by upregulating IRS1, IRS2 and AKT1, and a high glycolytic index, which indicate higher insulin sensitivity and activated glucose metabolism, suggesting that CD4+ cells of JAKi-treated patients regained insulin sensitivity. CD4+ cells of JAKi-treated patients displayed a decreased expression of cyclin-dependent kinase (CDK)1 and increased transcription of CDK inhibitors CDKN1A/p21 and CDKN2A/p16. This pattern was replicated in CD4+ cells treated with JAKi ex vivo and resulted in G1/S phase transition arrest, characterized by accumulation of cells in G1 phase and retention of Cell-Trace Violet, measured by flow cytometry. This was consequently supported by an increased expression of CDKN1A and CDKN2A genes. Flow cytometry analysis showed that insulin slowed down cell cycle by prolonging G2 phase. JAKi acted more potently by enhancing cell cycle arrest in every step of cell phase transition. In patients with hyperinsulinemia, the insulin-induced effect on cell cycle was seen in upregulation of CDKN2D gene and suppression of genes in the chromosomal passenger complex needed to accomplish mitosis.

Conclusion: This study shows that insulin has important immunosuppressive ability restricting activity of the effector Th1 cells. Hyperinsulinemia controls the adaptive immunity by suppressing IFNγ production and inducing senescence and in CD4 ⁺ T cells. Inhibition of JAK-STAT signaling enhances insulin sensitivity of CD4 ⁺ cells in RA patients.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.