Passive physical barrier modulates UVB-induced METosis-related MPO expression and activity, 25-hydroxyvitamin D3-1alpha-hydroxylase, and the shift of tissue-resident macrophages toward M1-associated iNOS

Abstract

Background: This study investigated the role of UVB radiation and the influence of a simulated passive barrier on the enzymatic conversion of 25-hydroxyvitamin D3 (25(OH)D₃) by 1-alpha hydroxylase and its effects on the functional activity of tissue-resident macrophages.

Methods: Murine peritoneal tissue-resident macrophages (PRMφs) were exposed to three conditions: (1) Baseline (Control group), with no light exposure; (2) UVB+/RF- group, exposed to UVB rays without passive barrier simulation; (3) UVB+/RF+ group, UVB exposure with a thin layer of rat fur to mimic the passive barrier on the skin.

Results: UVB exposure did not significantly alter 25OHD₃ levels across groups but led to a marked downregulation of 1-alpha hydroxylase, particularly with the simulated barrier. UVB slightly enhanced phagocytosis and significantly increased nitric oxide (NO) and hydrogen peroxide (H₂O₂) production. Moreover, hypochlorous acid (HOCl) levels were significantly upregulated in the UVB-exposed PRMφ group, whereas they returned to baseline levels in the UVB+/RF+ group. Furthermore, both MPO expression and activity were markedly upregulated after UVB exposure and downregulated in UVB+/RF+ group, suggesting that the overall effect of UVB on METosis-related MPO activity was substantially attenuated by the simulated barrier (for both comparisons, p < 0.001 by ANOVA test). Additionally, UVB exposure shifted PRMφs toward M1-phenotype, as evidenced by decreased ARG1 activity and increased iNOS activity and M1_(iNOS)-to-M2_(ARG1) ratio. Additionally, UVB downregulated catalase (CAT) activity and intracellular glucose (_iGLU) levels, with a stronger effect in the barrier group. While UVB increased total cellular cholesterol content (_tccCHOL), this effect was mitigated by the barrier. Finally, intracellular free calcium ion (_ifCa²⁺) levels remained unaffected by UVB but showed a slight increase with the barrier.

Conclusions: UVB exposure enhances tissue-resident macrophage function in a preclinical rat model, increasing respiratory burst, phagocytosis, and M1-like polarization. The simulated barrier modulates these effects, notably by reducing MPO expression and METosis-related activity, which suggests a potential attenuation of excessive inflammation. These findings provide valuable insights relevant to human immune modulation and support further translational research. Future studies should investigate the role of circadian rhythms and other cell types in UVB- and vitamin D-mediated immune modulation.

Keywords: 25-hydroxyvitamin D3-1alpha-hydroxylase; M1 macrophage-associated iNOS activity; METosis-related MPO expression and activity; UVB exposure; peritoneal tissue-resident macrophages; physical barrier simulation.

Figure 1

Flowchart of the current study. This study investigated the role of UVB radiation and the potential influence of passive physical barriers, such as simulated clothing, in preventing UVB exposure, on the enzymatic conversion of 25-hydroxyvitamin D3 (25(OH)D3) by 1-alpha hydroxylase, and assessed their effects on the phenotypic functional activities of tissue-resident macrophages. Experiments were conducted using murine peritoneal tissue-resident macrophages under three conditions: (i) Baseline (Control group), (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group).

Figure 2

Effects of UVB radiation and passive physical barrier on 25(OH)D₃ content of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). Levels of 25(OH)D₃ were measured using a chemiluminescent microparticle immunoassay. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). No significant differences were observed between groups using one-way ANOVA.

Figure 3

Effects of UVB radiation and passive physical barrier on 25-hydroxyvitamin D₃-1alpha-hydroxylase of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). The activity of 1α-hydroxylase assay was based on the two-point technique, determining the change in 25(OH)D₃ concentration over time and normalizing this change by the amount of protein in the sample and the time interval. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. ***p < 0.001.

Figure 4

Effects of UVB radiation and passive physical barrier on phagocytosis and respiratory burst activity of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). Phagocytosis activity was evaluated using the NBT method, based on its reduction to formazan by superoxide anions produced during the respiratory burst in phagocytic cells. Oxidative/respiratory burst was performed by measuring the levels of NO production, H₂O₂ levels, and HOCl. H₂O₂ levels were spectrophotometrically determined using a phenol red-based assay. NO production levels were spectrophotometrically determined using the sensitive colorimetric Griess method. HOCl levels were determined by measuring the decomposition of H₂O₂ through in vitro bromide oxidation by HOCl generated by activated cells in the presence of chloride released into the extracellular medium. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). H₂O₂: hydrogen peroxide, HOCL: hypochlorous acid, NBT: nitro-blue tetrazolium, NO: nitric oxide. Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Effects of UVB radiation and passive physical barrier on MPO expression and activity of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). MPO activity was quantified by directly measuring HOCl levels, normalized to total protein content, and expressed as percentage active chlorine per milligram of protein per one hour. MPO expression was assessed via immunofluorescence imaging, analyzed using CellProfiler software (v4.2.6, Broad Institute, USA). For imaging analysis, object dimensions were converted from pixels to micrometers (μm) to ensure accurate quantification. This conversion is based on an estimated image resolution of 0.58 μm per pixel, derived from an approximate field of view of 750 μm for an image width of 1296 pixels. The resolution was calculated as: $Resolution=\frac{Field of view width (\mu m) }{Image width (pixels)}= \frac{750 \mu m }{1296 pixels} \approx 0.5787\mu m/pixel$ . This estimation aligns with the theoretical resolution limit of the device (∼0.5 μm). To convert area values, the square of the resolution (0.58 μm/pixel)² was applied to pixel measurements, resulting in approximate areas in μm². For perimeter values, a linear conversion of 0.58 μm/pixel was applied. Therefore, the reported values should be considered approximate. The intensity measurements were normalized using a min-max scaling approach to ensure comparability across images with differing intensity ranges. This normalization method adjusts the raw pixel intensities (I) to a scale between 0 and 1, calculated as: $I_{normalized} =\frac{I- I_{min}}{I_{max}- I_{min}},$ where I_min and I_max represent the minimum and maximum intensity values in the dataset, respectively. This process preserves the relative differences between pixel intensities while standardizing their range aligning with the quantitative expectations of the theoretical resolution limits and ensuring that the observed patterns are not influenced by varying dynamic ranges of the original images. The normalization was performed independently for each group, allowing for direct comparisons of intensity distributions across experimental conditions. Finally, the weighted mean intensity (WMI) values were calculated to account for the normalized intensity distributions across the analyzed regions of interest (ROIs). These values were derived as: $WMI=\frac{ {\sum }_{i }{w}_i{ }_{\times I_i} }{{\sum }_{i }{w}_i}$ , where I_i represents the intensity value of a given pixel, and W denotes the pixel area. This method ensures that the calculated intensity reflects the contribution of each pixel proportionally to its area within the ROI. Overlaid histograms representing the fluorescence intensity distribution for the three experimental groups. Intensities were normalized to ensure comparability, and the x-axis represents fluorescence intensities (a.u.), while the y-axis indicates the pixel count. For histogram data, values are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). HOCl: hypochlorous acid, MPO: myeloperoxidase, RF: rat fur, UVB: ultraviolet B. Significant differences are indicated by an asterisk. Statistical analyses were performed using the Kruskal-Wallis test for non-normally distributed data (area and perimeter) or one-way ANOVA for normally distributed data (normalized fluorescence intensity, normalized weighted mean intensity, and MPO activity). **p < 0.01, ***p < 0.001.

Figure 6

Effects of UVB radiation and passive physical barrier on the M1/M2 dichotomy. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). The M1_(iNOS)/M2_(ARG1) dichotomy was determined mathematically by measuring the M1-to-M2 ratio. M1 activity was determined by measuring iNOS (EC 1.14.13.39) activity through the quantification of NO production normalized to protein content, whereas M2 activity was assessed by measuring the amount of urea generated by ARG1 (EC 3.5.3.1). Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). iNOS: inducible nitric oxide synthase, ARG1: arginase 1. Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. **p < 0.01.

Figure 7

Effects of UVB radiation and passive physical barrier on catalase-based cell protection activity of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). CAT (EC 1.11.1.6)-based cell protection activity was spectrophotometrically assessed by measuring H₂O₂ decomposition through titanium sulfate-based detection. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). CAT: catalase activity, H₂O₂: hydrogen peroxide. Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. ***p < 0.001.

Figure 8

Effects of UVB radiation and passive physical barrier on trained immunity activation-based _tccCHOL signature of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). Trained immunity activation-based _tccCHOL signature was measured spectrophotometrically through Trinder’s reaction. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). _tccCHOL: total cellular cholesterol content. Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. *p < 0.05.

Figure 9

Effects of UVB radiation and passive physical barrier on _iGLU-based immune cell metabolism of PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). _iGLU-based immune cell metabolism was assessed spectrophotometrically by measuring H₂O₂ produced during glucose oxidation by glucose oxidase. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). _iGLU: intracellular glucose. Significant differences are indicated by an asterisk. Statistical analyses were performed using one-way ANOVA. ***p < 0.001.

Figure 10

Effects of UVB radiation and passive physical barrier on free Ca²⁺ levels in PRMφs. Experiments were conducted on murine peritoneal tissue-resident macrophages (PRMφs) under three conditions: (i) Control group, (ii) Controlled exposure to UVB rays (UVB+ group), and (iii) UVB exposure with a simulated physical barrier (RF+ group). _ifCa²⁺ levels were assessed using the ortho-cresolphthalein complexone (oCPC) method. Data are presented as mean ± standard error of the mean (SEM) from four independent repetitions (n = 12 per group). No significant differences were observed between groups using one-way ANOVA. _ifCa²⁺, intracellular free calcium ions.