Proton irradiation orchestrates macrophage reprogramming through NFκB signaling

Abstract

Tumor-associated macrophages (TAMs) represent potential targets for anticancer treatments as these cells play critical roles in tumor progression and frequently antagonize the response to treatments. TAMs are usually associated to an M2-like phenotype, characterized by anti-inflammatory and protumoral properties. This phenotype contrasts with the M1-like macrophages, which exhibits proinflammatory, phagocytic, and antitumoral functions. As macrophages hold a high plasticity, strategies to orchestrate the reprogramming of M2-like TAMs towards a M1 antitumor phenotype offer potential therapeutic benefits. One of the most used anticancer treatments is the conventional X-ray radiotherapy (RT), but this therapy failed to reprogram TAMs towards an M1 phenotype. While protontherapy is more and more used in clinic to circumvent the side effects of conventional RT, the effects of proton irradiation on macrophages have not been investigated yet. Here we showed that M1 macrophages (THP-1 cell line) were more resistant to proton irradiation than unpolarized (M0) and M2 macrophages, which correlated with differential DNA damage detection. Moreover, proton irradiation-induced macrophage reprogramming from M2 to a mixed M1/M2 phenotype. This reprogramming required the nuclear translocation of NFκB p65 subunit as the inhibition of IκBα phosphorylation completely reverted the macrophage re-education. Altogether, the results suggest that proton irradiation promotes NFκB-mediated macrophage polarization towards M1 and opens new perspectives for macrophage targeting with charged particle therapy.

Fig. 1. Schematic outline of the irradiation procedure for macrophages.

THP-1 monocytes were differentiated into macrophages (M0) with 150 nM PMA in cloning cylinder, placed at the center of the irradiation chamber. Macrophages were polarized in M1 phenotype with 10 pg/ml LPS and 20 ng/ml IFN-γ during 24 h incubation or were polarized in M2 phenotype with 20 ng/ml IL-4 and IL-13 during 48 h incubation. THP-1 monocytes were differentiated on the appropriate day, in order to obtain the three phenotypes on day 4. Following the experiment that was performed, the biological material was collected 8, 12, 16, or 24 h after proton or X-ray irradiation

Fig. 2. Early radioresistance of M1 macrophages after moderate doses of proton irradiation.

M0, M1, and M2 macrophages were irradiated with different doses of protons. Viability was assessed by ethidium bromide−acridin orange at different times postirradiation. a Representative ethidium bromide−acridine orange staining images of M1 and M2 macrophages 16 h after proton irradiation (0 Gy and 10 Gy). Lived cells appeared in green while dead cells are stained in orange. b Quantification of viability (%) in M0, M1, and M2 macrophages, 8 or 16 h after proton irradiation. N = 3 for each dose (mean ± SD). One-way ANOVA analyses followed by Dunnett’s multiple comparisons post-tests were performed on data; *p ≤ 0.05; **p < 0.01; ***p < 0.001

Fig. 3. M1 resistance to proton irradiation correlates with a higher γH₂AX and 53BP1 labeling.

M0, M1, and M2 macrophages were irradiated with different doses of protons. The evaluation of DNA damage following proton irradiation was performed by phosphorylated H₂AX (γH₂AX) or 53BP1 labeling. a Representative immunofluorescence labeling of γH₂AX for M0, M1, and M2 macrophages 15 min after proton irradiation. γH₂AX labeling appears in green and nuclei are stained in blue (To-pro). b Quantification of the mean γH₂AX intensity per nucleus 15 min after proton irradiation. Results are expressed in mean pixel intensity value and are normalized to the nonirradiated condition (fold change). Quantifications were performed on minimum five images per condition; representative experiment (N = 3, mean ± SD). c Mean γH₂AX intensity after proton irradiation (3 Gy), several times postirradiation. Each point corresponds to the mean γH₂AX intensity at time t (Tt) normalized to time 0 (T0). Quantifications were performed on minimum ten images per condition; representative experiment, N = 3 (mean ± SD). One-way ANOVA analyses were performed on data, followed by Dunnett’s post-tests; *p ≤ 0.05; **p < 0.01; ***p < 0.001. d Representative immunofluorescence labeling of 53BP1 for M0, M1, and M2 macrophages 15 min after proton irradiation. 53BP1 labeling appears in green and nuclei are stained in blue (To-pro). e Quantification of the 53BP1 intensity per nucleus after proton irradiation (3 Gy), several times postirradiation. Each point corresponds to the mean 53BP1 intensity at time t (Tt), normalized to time 0 (T0). Quantifications were performed on five images per condition; N = 1 (mean ± SD). One-way ANOVA analyses were performed on data, followed by Dunnett’s post-tests; *p ≤ 0.05; **p < 0.01; ***p < 0.001

Fig. 4. Proton irradiation induces macrophage reprogramming.

M0, M1, and M2 macrophages were irradiated with different doses of protons (0, 5, and 10 Gy). a 24 h after proton irradiation, mRNA levels of M1 (TNFα, IL-6, IL-8) and M2 (CCL22, IL-10, EGF) markers were assessed by RT-qPCR (N = 3, mean ± SD). One-way ANOVA analyses followed by Dunnett’s multiple comparison tests were performed to evaluate the significance (*p ≤ 0.05; **p < 0.01; ***p < 0.001). b 24 h after proton irradiation, TNFα, IL-8, and IL-6 secretion was evaluated by ELISA. Results are expressed in pg/ng of proteins and are normalized to the nonirradiated condition (fold change) for each macrophage phenotype; ND was used for not detected (N = 3, mean ± SD). One-way ANOVA analyses followed by Kruskal−Wallis multiple comparison tests were performed on data (*p ≤ 0.05; **p < 0.01; ***p < 0.001)

Fig. 5. Proton irradiation induces nuclear translocation of p65 (NFκB).

M0, M1, and M2 macrophages were irradiated by different doses of protons. 2 h after the irradiation, the nuclear translocation of p65 was evaluated by NFκB p65 immunofluorescence labeling. NFκB p65 is stained in green and nucleus appears in blue. a Nuclear translocation of NFκB p65 is indicated (arrow) on representative immunofluorescence labeling images for M0, M1, and M2 macrophages after irradiation. b Quantification of the mean NFκB p65 intensity per nucleus 2 h after proton irradiation. Results are expressed in percentage of cells with nuclear NFκB p65. Quantifications were performed on minimum five images per condition (N = 3, mean ± SD). An unpaired t test was performed on data (*p ≤ 0.05; **p < 0.01; ***p < 0.001)

Fig. 6. NFκB inhibition reverts proton irradiation-induced macrophage reprogramming.

M0 and M2 macrophages were irradiated (IR) with protons (10 Gy) with or without NFκB inhibitor (Bay 11-7082—5 μM). The inhibitor was added 1 h before irradiation for the following 12 h. Macrophage polarization was evaluated by RT-qPCR 12 h after proton irradiation by the analysis of M1 (TNFα, IL-6, IL-8) and M2 (CCL22, IL-10, EGF) marker mRNA level in (a) M0 macrophages (N = 3, mean ± SD) and b M2 macrophages (N = 4, mean ± SD). Two-way ANOVA analyses followed by Tukey’s multiple comparison tests were performed on data; *p ≤ 0.05; **p < 0.01; ***p < 0.001