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

Abstract

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.

Microbiota metabolite butyrate constrains neutrophil functions and ameliorates mucosal inflammation in inflammatory bowel disease

“…15 Moreover, our recent work also demonstrated a novel effect of cyclosporine A on neutrophils, showing that cyclosporine A functions as fueling glycolysis and aerobic oxidation to restrain excessive activation, contributing to the induction of clinical remission in acute severe UC patients. 35 In contrast, inflamed mucosa from patients with active IBD is infiltrated with large numbers of neutrophils, and the degree of neutrophil infiltration is thought to be instrumental for the assessment of disease activity. 17 Neutrophil-derived cytokines and chemokines play an important role in the initiation and perpetuation of inflammation.…”

GPR65 promotes intestinal mucosal Th1 and Th17 cell differentiation and gut inflammation through downregulating NUAK2

“…qRT‐PCR was accomplished as depicted formerly. 29 , 30 All primer sequences were listed as indicated in Table S2 .…”

“…Immunoblotting was performed as depicted formerly. 29 , 31 Splenic naïve CD4 + T cells were obtained from Gpr65 flx/flx and Gpr65 ΔCD4 mice. The primary antibodies including anti‐GPR65 (1:200, Invitrogen), anti‐PKA (1:1000, Cell Signalling Technologies), anti‐C‐Raf (1:750, Cell Signalling Technologies), anti‐P‐C‐Raf (1:1000, Cell Signalling Technologies), anti‐ERK1/2 (1:500, Invitrogen), anti‐LKB1 (1:500, Cell Signalling Technologies), anti‐NUAK2 (1:500, Invitrogen) and anti‐Actin (1:3000, Santa Cruz Biotechnology) were used.…”

“…Immunoblotting was performed as depicted formerly 29,31 . Splenic naïve CD4 + T cells were obtained from Gpr65 flx/flx and Gpr65 ΔCD4 mice.…”

TOB1 Blocks Intestinal Mucosal Inflammation Through Inducing ID2-Mediated Suppression of Th1/Th17 Cell Immune Responses in IBD

“…PBMC were separated from EDTA anticoagulated PB of IBD patients and healthy donors by Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) gradient centrifugation. 38 CD4 + T cells were then isolated from PBMCs using anti-human CD4 magnetic beads (BD Biosciences, San Diego, CA, USA) and cultured in complete RPMI-1640 medium under stimulation with immobilized anti-human CD3 mAb (5 μg/mL) and soluble anti-human CD28 mAb (2 μg/mL) (eBioscience, San Diego, CA) for 48 hours. 39 These preactivated CD4 + T cells were reseeded in 24-well plates at a density of 1 × 10 5 /well, and added with LV-TOB1, LV-shTOB1, LV-ID2, LV-shID2, and LV-NC, respectively (multiplicity of infection = 180), followed by centrifugation at 800 g for 2 hours at 37°C.…”