5-Fluorouracil disrupts skeletal muscle immune cells and impairs skeletal muscle repair and remodeling

Abstract

5-Fluorouracil (5FU) remains a first-line chemotherapeutic for several cancers despite its established adverse side effects. Reduced blood counts with cytotoxic chemotherapies not only expose patients to infection and fatigue, but can disrupt tissue repair and remodeling, leading to lasting functional deficits. We sought to characterize the impact of 5FU-induced leukopenia on skeletal muscle in the context of remodeling. First, C57BL/6 mice were subjected to multiple dosing cycles of 5FU and skeletal muscle immune cells were assessed. Second, mice given 1 cycle of 5FU were subjected to 1.2% BaCl₂ intramuscularly to induce muscle damage. One cycle of 5FU induced significant body weight loss, but only three dosing cycles of 5FU induced skeletal muscle mass loss. One cycle of 5FU reduced skeletal muscle CD45⁺ immune cells with a particular loss of infiltrating CD11b⁺Ly6c^Hi monocytes. Although CD45⁺ cells returned following three cycles, CD11b⁺CD68⁺ macrophages were reduced with three cycles and remained suppressed at 1 mo following 5FU administration. One cycle of 5FU blocked the increase in CD45⁺ immune cells 4 days following BaCl₂; however, there was a dramatic increase in CD11b⁺Ly6g⁺ neutrophils and a loss of CD11b⁺Ly6c^Hi monocytes in damaged muscle with 5FU compared with PBS. These perturbations resulted in increased collagen production 14 and 28 days following BaCl₂ and a reduction in centralized nuclei and myofibrillar cross-sectional area compared with PBS. Together, these results demonstrate that cytotoxic 5FU impairs muscle damage repair and remodeling concomitant with a loss of immune cells that persists beyond the cessation of treatment.NEW & NOTEWORTHY We examined the common chemotherapeutic 5-fluorouracil's (5FU) impact on skeletal muscle immune cells and skeletal muscle repair. 5FU monotherapy decreased body weight and muscle mass, and perturbed skeletal muscle immune cells. In addition, 5FU decreased skeletal muscle immune cells and impaired infiltration following damage contributing to disrupted muscle repair. Our results demonstrate 5FU's impact on skeletal muscle and provide a potential explanation for why some patients may be unable to properly repair damaged tissue.

Keywords: chemotherapy; macrophages; monocytes; muscle repair; skeletal muscle.

Graphical abstract

Figure 1.

Experimental design and animal characteristics. A: 5-fluorouracil (5FU) was solubilized in phosphate-buffered saline at 3.5 mg/mL and administered to the mice at 35 mg/kg via intraperitoneal injection once daily for 5 days (1 cycle), 9 day rest and five additional daily injections, another 9 day rest, with a final five injections (3 cycles) and allowed to recover for 30 days (Recovery). PBS controls were taken with each time point (n = 4/group—12). B: relative body weight change shown as the % change from day 0 to the end of each treatment period. C: daily food intake change shown as the % change from day 0 to the end of each treatment period. D: select hindlimb muscle weights given in milligrams (mg) at euthanasia from each group. E: gonadal fat pad weight given in mg at euthanasia from each group. F: select organ weights in mg at euthanasia from each group. PBS n = 12, one cycle n = 8, three cycles n = 8, and Recovery n = 8; n represents number of animals. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. One-way ANOVA.

Figure 2.

The effects of 5-fluorouracil (5FU) on skeletal muscle immune cells. Cells were gated for nondebris (SSC-A × FSC-A), FSC singlets (FSC-W × FSC-H), SSC singlets (SSC-W × SSC-H), and Live ZombieGreen^−/Low. A: live single cells were then gated with SSC-A and CD45 for immune cells and the relative abundance of CD45⁺ cells as % of live cells were graphed. B: CD45⁺ immune cells were then gated with CD11b and Ly6c and the relative abundance of CD11b⁺Ly6c^Lo/Int/Hi as a % of CD45⁺ immune cells were graphed. C: CD45⁺ immune cells were then gated with CD11b and CD68 for total macrophages and the relative abundance of CD11b⁺CD68⁺ as a % of CD45⁺ immune cells were graphed. D: CD11b⁺CD68⁺ macrophages were gated with CD11c and CD206. CD11c⁻CD206⁻ cells were considered M0-like macrophages, CD11c⁺CD206⁻ cells were considered M1-like macrophages, CD11c⁻CD206⁺ cells were considered M2-like macrophages, and CD11c⁺CD206⁺ cells were considered M1-M2-like transitional macrophages. The relative abundance of each cell subset as a % of CD11b⁺CD68⁺ macrophages were graphed. PBS n = 12, one cycle n = 8, three cycles n = 8, and Recovery n = 8; n represents number of animals. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. Two-way ANOVA.

Figure 3.

The impact of 5-fluorouracil (5FU) on skeletal muscle macrophages following BaCl₂-induced muscle damage. A: experiment 2 experimental design. 5FU was solubilized in phosphate-buffered saline at 3.5 mg/mL and administered to the mice at 35 mg/kg via intraperitoneal injection once daily for 5 days. Mice were then subjected to intramuscular injection of 1.2% barium chloride (BaCl₂) and tissue was collected at 18 h, 96 h, 14 days, and 28 days following BaCl₂. B: transmitted electron microscopy (TEM) images taken at 96 h following BaCl₂ in mice given PBS or 5FU to verify muscle damage. Scale bar = 600 nm. C: single live cells isolated from undamaged (und) 18 hours (h) post BaCl₂ and 96 h post BaCl₂ were gated with SSC-A and CD45 for immune cells. D: CD45⁺ immune cells were then gated with CD11b and CD68 for total macrophages. E: CD11b⁺CD68⁺ macrophages were gated with CD11c and CD206 to examine macrophage polarization. CD11c⁻CD206⁻ cells were considered M0-like macrophages, CD11c⁺CD206⁻ cells were considered M1-like macrophages, CD11c⁻CD206⁺ cells were considered M2-like macrophages, and CD11c⁺CD206⁺ cells were considered M1-M2-like transitional macrophages. F: the relative abundance of CD45⁺ cells as % of live cells. G: the relative abundance of CD11b⁺CD68⁺ as a % of CD45⁺ immune cells. H–K: the relative abundance of each macrophages cell subset as a % of CD11b⁺CD68⁺ macrophages. PBS undamaged n = 4, 5FU undamaged n = 6, PBS at 18 h n = 4, 5FU at 18 h n = 4, PBS at 96 h n = 4, 5FU at 96 h n = 6. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. $Significant main effect of damage. Two-way ANOVA.

Figure 4.

The impact of 5-fluorouracil (5FU) on skeletal muscle monocyte and neutrophil infiltration following BaCl₂-induced muscle damage. A: single live CD45⁺ immune cells isolated from undamaged (und) 18 hours (h) post BaCl₂ and 96 h post BaCl₂ were gated with CD11b and Ly6g for neutrophils. B: the relative abundance of CD11b⁺Ly6g⁺ neutrophils as a % of CD45⁺ immune cells were graphed. C: similarly live CD45⁺ immune cells were gated with CD11b and Ly6c for infiltrating monocytes. D–F: the relative abundance of CD11b⁺Ly6c^Lo/Int/Hi as a % of CD45⁺ immune cells were graphed. PBS undamaged n = 4, 5FU undamaged n = 6, PBS at 18 h n = 4, 5FU at 18 h n = 4, PBS at 96 h n = 4, 5FU at 96 h n = 6; n represents number of animals. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. Two-way ANOVA.

Figure 5.

The impact of 5-fluorouracil (5FU) on skeletal muscle regeneration and remodeling 14 days following BaCl₂-induced muscle damage. A: representative hematoxylin and eosin (H&E) stain rectus femoris muscles from control and damaged skeletal muscle in mice given 5FU or PBS 14 days after insult. Scale bars = 50 µm. B: number of centralized nuclei per 100 fibers measured. col3a1 (C) and col1a1 (D) gene expression in the rectus femoris 14 days following BaCl₂. Data were normalized to PBS undamaged. E: representative immunofluorescent ×40 images of wheat germ agglutinin (WGA), macrophages (F4/80), and nuclei (DAPI) in rectus femoris muscle from control and damaged skeletal muscle in mice given 5FU or PBS 14 days after insult. Scale bar = 50 µm. PBS undamaged n = 7, 5FU undamaged n = 7, PBS 14 at days n = 7, 5FU at 14 days n = 7. n = 3/group histology and n = 4/group gene expression; n represents number of animals. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. $Significant main effect of damage. Two-way ANOVA.

Figure 6.

The impact of 5-fluorouracil (5FU) on skeletal muscle regeneration and remodeling 28 days following BaCl₂-induced muscle damage. A: representative hematoxylin and eosin (H&E) stain rectus femoris muscles from control and damaged skeletal muscle in mice given 5FU or PBS 28 days after insult. ×20 scale bar = 100 µm, ×40 scale bar = 50 µm. B: number of centralized nuclei per 100 fibers measured. C: fiber size (µm²) distribution in all groups. D: mean myofibrillar cross-sectional area (mCSA) 28 days following BaCl₂. col3a1 (E) and col1a1 (F) gene expression in the rectus femoris 28 days following BaCl₂. Data were normalized to PBS undamaged. PBS undamaged n = 3, 5FU undamaged n = 3, PBS at 28 days n = 3, 5FU at 28 days n = 3; n represents number of animals. Significance was set at P < 0.05. Different letter indicates significant different groups when an interaction was observed. Two-way ANOVA.

Figure 7.

Representative scanning electron micrographs at ×1,000 magnification 14 days following BaCl₂-induced muscle damage. A proximal portion of the rectus femoris was subjected to scanning electron microscopy (SEM). Muscles were nonspecifically searched for adjacent parallel fibers to examine the extracellular matrix and collagen fibers. Images were collected at ×1.000. Scale bar = 10 μm. 5FU, 5-fluorouracil; Und, undamaged.

Figure 8.

Representative scanning electron microscopy images at ×5,000 magnification 14 days following BaCl₂-induced muscle damage. A proximal portion of the rectus femoris was subjected to SEM. Muscles were nonspecifically searched for adjacent parallel fibers to examine the extracellular matrix and collagen fibers. Images were collected at ×5,000. Scale bar = 5 μm. 5FU, 5-fluorouracil; SEM, scanning electron microscopy; Und, undamaged.