Influence of Different Nicotine Sources on Exercise-Driven Immune Responses of Peripheral Blood Monocytes

Abstract

Tobacco smoking is closely associated with pro-inflammatory immunological alterations, whereas regular physical exercise is well known to lower systemic inflammations and related immune cell activities. The combined effects of smoking, nicotine pouch use, vaping, and exercise on individual immunological responses remain incompletely understood, especially in view of alternative nicotine delivery systems. In this study, we analyzed the immediate impact of different nicotine sources on exercise monocyte subsets in 16 human subjects using a four-arm cross-over design. Distribution of circulating CD14/CD16 monocyte subsets and expression of the monocytic checkpoint molecule PD-L1 (programmed cell death ligand-1) were analysed via whole blood flow cytometry measurements. Plasma cytokines were evaluated using membrane-based cytokine arrays and enzyme-linked immunosorbent assays (ELISA). Data revealed significant distributions of circulating monocytes subsets in response to nicotine consumption and physical stress. In contrast, exercise-driven increased monocytic PD-L1 was clearly attenuated following the consumption various nicotine delivery systems. Furthermore, significantly increased plasma growth hormone levels were detected in response to physical stress in combination with cigarette consumption. Our data clearly illustrates a significant influence of nicotine consumption on the cellular characteristics of circulating monocyte subsets and on proper exercise-driven immune responses within a short period of time, which makes the widespread trivialization of alternative nicotine sources questionable.

Keywords: PD-L1; exercise; growth hormone; monocytes; smoking.

Figure 1

Flowchart and study design for each participant. Every device is tested once, whereas the order is randomly determined at the beginning of each measurement day (Cig—smoking a combustible cigarette; E-Cig—vaping an e-cigarette with tobacco flavor; Pouch—consumption of a nicotine pouch; Sham—vaping an e-cigarette without liquid as a control).

Figure 2

Study scheme and flow cytometric analysis of peripheral blood monocyte subsets. (A) Representative example gating scheme of monocyte subset analysis with regard to the forward scatter (FSC)/sideward scatter (SSC) characteristics and the CD14/CD16 expression levels of circulating classical (CM), intermediate (IM) and non-classical monocytes (NCM). (B) Study scheme illustrates the measuring points (1) before nicotine consumption, (2) after nicotine consumption, (3) after physical stress, and (4) after 30 min break. Measuring point 2 can be distinguished in (A) control, (B) cigarette, (C) e-cigarette, and (D) nicotine pouch.

Figure 3

Flow cytometric analysis of peripheral blood monocyte subsets. Percentages of circulating classical (CM), intermediate (IM) and non-classical monocytes (NCM) in the peripheral blood of our cohort during the course of our study (pre; nicotine consumption; physical stress; post). The different parameters (A) control (consumption of nicotine free e-cigarette), (B) cigarette, (C) e-cigarette, and (D) pouch where investigated with regard to the measuring point ‘nicotine consumption’. *: p < 0.05; **: p < 0.01.

Figure 4

Checkpoint molecule PD-L1 on peripheral blood monocyte subsets (CM: classical monocytes; IM: intermediate monocytes; NCM: non-classical monocytes) during the course of our study (pre; nicotine consumption; physical stress; post). Different parameters (A) control (consumption of nicotine free e-cigarette), (B) cigarette, (C) e-cigarette, and (D) pouch were investigated with regard to the measuring point ‘nicotine consumption’, respectively. *: p < 0.05. MFI: mean fluorescence intensity.

Figure 5

Cytokine screening during the course of our study. (A) Raw images of membrane-based cytokine arrays of plasma samples of one tested individual revealed increased growth hormone levels in response to cigarette smoking and physical stress at measuring point 3 compared to the non-smoking control. (B) Semiquantitative analysis was conducted by measuring the density of the growth hormone dots. (C) ELISA measurements of growth hormone (GH, pg/mL) plasma concentrations during the course of our study. Data revealed significantly increased GH levels in response to physical stress in combination with the consumption of the different nicotine sources such as cigarette, e-cigarette, and pouch (measuring point 3) compared to the control (consumption of nicotine free e-cigarette). *: p < 0.05; **: p < 0.01; ***: p < 0.001.