Cyclosporine modulates neutrophil functions via the SIRT6-HIF-1α-glycolysis axis to alleviate severe ulcerative colitis.

BACKGROUND:Cyclosporine A (CsA) is routinely used to treat patients with steroid-refractory acute severe ulcerative colitis (ASUC). Here, we studied the underlying mechanisms of CsA-mediated alleviation in ASUC patients. METHODS:Neutrophil functions including expression of cytokines, apoptosis, and migration were measured by qRT-PCR, flow cytometry, and Transwell assay. Dynamic changes of glycolysis and tricarboxylic acid (TCA) cycle were measured by a Seahorse extracellular flux analyzer. Gene differences were determined and verified by RNA sequencing, qRT-PCR, and Western blotting. Small interfering RNA and inhibitors were used to knock down Sirtuin 6 (SIRT6) in HL-60 cells and block expression of SIRT6, hypoxia-inducible factor-1α (HIF-1α), and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) in neutrophils. RESULTS:We found that HIF-1α expression and glycolysis significantly increased, while the release of IL-8, myeloperoxidase (MPO) and reactive oxygen species (ROS), the apoptosis, and ability of migration markedly decreased in neutrophils of ASUC patients who responded to CsA (Response group) compared with those who did not respond to CsA (Nonresponse group). We also observed that CsA-induced functional alternation of neutrophils was initiated through suppressing SIRT6 expression, which is responsible for expression of the downstream signaling molecules (e.g., HIF-1α, PFKFB3) and PDK4 ubiquitination, leading to fueling neutrophil glycolysis and TCA cycle. Furthermore, blockage of SIRT6 signaling demonstrated to be the same functional changes as CsA to decrease the migration of neutrophils. CONCLUSIONS:The data reveal a novel mechanism of CsA in alleviating ASUC by promoting neutrophil HIF-1α expression and restricting excessive neutrophil activation in a SIRT6-HIF-1α-glycolysis axis, suggesting SIRT6 as a candidate target for maintaining mucosal homeostasis and treating intestinal inflammation.