Differential effect of intermittent hypoxia and sleep fragmentation on PD-1/PD-L1 upregulation

Abstract

Immunosurveillance is compromised in patients with obstructive sleep apnea (OSA) as reflected by overexpression of the programmed death cell receptor and its ligand (PD-1/PD-L1) coinhibitory axis. However, the contributions of intermittent hypoxia (IH) and sleep fragmentation (SF) are unclear. We therefore evaluated the expression of PD-1 and PD-L1 on immune cells from mice subjected to IH or SF, and in human cells exposed to IH, oxidative stress, or both conditions. Six-week-old male C57BL/6J mice were exposed to either IH or SF using previously established in vivo models. Moreover, human peripheral blood mononuclear cells (PBMC) were cultured overnight under normoxia, IH, hydrogen peroxide (H2O2), or both. Murine splenocytes and human PBMC were isolated, and labeled using surface-specific antibodies for flow cytometry analysis. Compared to control mice, IH induced higher expression of PD-L1 on F4/80 cells and of PD-1 on CD4+ and CD8+ T-cells, whereas no significant changes emerged after SF. In vitro models of IH and oxidative stress showed similar changes for expression of PD-L1 on human monocytes and PD-1 on CD4+ T-cells. Furthermore, H2O2 increased PD-1 expression on CD8+ T-cells, compromising their cytotoxic capacity assessed by perforin expression, similar to IH. No evidence of synergistic effects was apparent. Therefore, PD-1/PD-L1 upregulation reported in patients with OSA appears to be preferentially mediated by IH rather than SF.

SMAD4 Overexpression in Patients with Sleep Apnoea May Be Associated with Cardiometabolic Comorbidities

“…In order to verify the role of HIF1α in this context, we used an in vitro model of IH with monocytes from healthy subjects as we have previously described [ 21 ]. Additionally, we treated HV monocytes with PX-478 ( S -2-amino-3-[4′- N , N ,-bis(2-chloroethyl)amino]phenyl propionic acid N -oxide dihydrochloride) to decrease HIF1α expression [ 32 , 33 , 34 ].…”

“…Intermittent hypoxia (IH) models were performed using an incubator chamber attached to an external O 2 /N 2 computer-driven controller using BioSpherix OxyCycler-42 (Redfield, NY, USA). This strategy mimicked the cyclical changes in O 2 concentrations that control air gas levels, maintaining CO 2 as previously described in detail [21]. Briefly, the IH model cycled O 2 saturation in the medium at 1% for 2 min, followed by 20% for 10 min, with CO 2 maintained at 5%.…”

“…In OSA patients, SMAD4 mRNA expression was significantly associated both with HIF1α (r = 0.4100, p = 0.0095) and mTOR mRNA expression (r = 0.3304 and p = 0.0373) (Figure 2A,B). In order to verify the role of HIF1α in this context, we used an in vitro model of IH with monocytes from healthy subjects as we have previously described [21]. Additionally, we treated HV monocytes with PX-478 (S-2-amino-3-[4′-N,N,-bis(2-chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) to decrease HIF1α expression [32][33][34].…”

“…Additionally, intermittent hypoxia (IH) triggers the hypoxia inducible factor (HIF1α) in OSA patients, in in vitro and in animal models [ 6 , 21 , 22 ]. Moreover, HIF1α expression induces TGFβ expression, as we have previously reported in OSA patients [ 6 ].…”

Obesity attenuates the effect of sleep apnea on active TGF-ß1 levels and tumor aggressiveness in patients with melanoma

“…Cell culture. C81-61 human cutaneous melanoma cell line 49 was cultured under normoxia or intermittent hypoxia (IH) conditions over night, as previously described 50 . The culture medium was supplemented with human leptin (230-30112-10, RayBiotech) at either a concentration similar to that detected in patients or two- www.nature.com/scientificreports/ fold that level (5 and 10 ng mL −1 , respectively).…”