ROS-activated CXCR2+ neutrophils recruited by CXCL1 delay denervated skeletal muscle atrophy and undergo P53-mediated apoptosis

Peripheral nerve injury causes slow reinnervation and prolonged dysfunction of denervated skeletal muscle. While inflammatory mediators are detected in denervated muscle, it is unclear whether neutrophils infiltrate indirectly damaged muscle and contribute to inflammation and atrophy dynamics. The study asks: Do neutrophils infiltrate denervated muscle lacking direct injury? What signals activate and recruit them? What is their functional role in muscle atrophy, and how are they resolved? Prior work implicates ROS in neutrophil activation and denervation-induced muscle oxidative stress, suggesting a potential ROS–neutrophil link. Given neutrophil heterogeneity and context-specific chemokine cues, the authors hypothesize that ROS-activated neutrophil subsets (notably CXCR2+ cells recruited by CXCL1) infiltrate denervated muscle, delay early atrophy, and subsequently undergo P53-mediated apoptosis to resolve inflammation.

- Muscle-resident macrophages maintain homeostasis and support regeneration; during inflammation they produce proinflammatory mediators. Neutrophils, absent from normal muscle, rapidly infiltrate directly injured tissues to mediate acute inflammation and repair. - ROS generation primes/activates neutrophils during infectious inflammation and is implicated in denervation-induced atrophy and systemic stress from ischemia/hypoxia after denervation. - Neutrophils exhibit functional heterogeneity; subsets (e.g., CXCR2+ vs. CCR2+) respond to distinct chemokines, implying that nonselective modulation of neutrophil numbers can be harmful. - Resolution of inflammation requires timely neutrophil apoptosis; mediators can prolong or accelerate neutrophil lifespan. P53 is a canonical regulator of apoptosis and has roles in muscle catabolic pathways. These works motivate dissecting which neutrophil subsets, chemokines, and apoptosis pathways operate in denervated muscle, an aseptic, indirectly injured context.

- Animals: Male C57BL/6 (N=137), Ly6g-DTR-GFP (N=6), and P53 knockout (N=18) mice (6–8 weeks, 22–25 g). Unilateral sciatic nerve transection was performed to induce denervation. Mice were randomized to time points and treatments. IACUC-approved. - Intravital imaging (SD-IVM): Gastrocnemius muscle was exposed under isoflurane. A Zeiss Axio Observer Z1 with Yokogawa CSU-X spinning disk and EMCCD camera acquired images (10x/25x). PECAM-1 (i.v.) labeled microvasculature; topical anti-Ly6G labeled neutrophils; FITC-albumin assessed vascular patency/permeability. - 3D tissue imaging: Gastrocnemius muscles were fixed, paraffin-embedded, serially sectioned (50 slices, 6 μm), double immunostained, whole-slide scanned, and reconstructed with voloom/Imaris for 3D visualization of neutrophils relative to vessels. - Flow cytometry: Single-cell suspensions from gastrocnemius and peripheral blood were prepared. Cells were stained with FVS viability dye and antibodies against CD45, Ly6G, CD11b, CXCR2 (CD182), and CCR2 (CD192). TUNEL assays were performed for apoptosis on permeabilized cells. Data analyzed with FlowJo. - ROS measurement and inhibition: Cellular ROS assessed with DCFH-DA (10 μM) by flow cytometry. Mice received L-NAC (1.1 mmol/kg/day, i.v. tail vein) for 3 days (including denervation day) or saline control. - RNA-seq: Total RNA from muscle was extracted; libraries prepared with TruSeq Stranded mRNA; sequenced on Illumina HiSeq X Ten. QC (Trimmomatic), alignment (hisat2), quantification (cufflinks, htseq-count), DEGs (DESeq; P<0.05; fold change >2 or <0.5), GO/KEGG enrichment in R. - qPCR: RNA from gastrocnemius used for cDNA synthesis; qPCR measured CXCL1 normalized to GAPDH with specified primers. - Neutrophil depletion: Anti-Ly6G (1A8) i.p. (400 μg initial then 100 μg thrice weekly) in WT mice; Ly6g-DTR-GFP mice received diphtheria toxin (10 μg/g i.p., thrice weekly) for depletion. - Muscle atrophy assays: Gastrocnemius wet weight measured at 7 and 14 days; percent loss relative to contralateral side. HE staining quantified myofiber area, diameter, and density by blinded analysis. - Statistics: Mean ± SEM; t tests for two-group comparisons; one-way ANOVA for multiple groups; Friedman test for repeated measures across time; significance at P<0.05.

- Neutrophil dynamics after denervation: - Flow cytometry showed sustained early increases in neutrophils in peripheral blood and denervated gastrocnemius, peaking at 12 h post-denervation, then declining to a lower level by day 3 (n=4/group; P<0.05 or P<0.01 vs 0 h). - ROS elevation and role in neutrophil activation: - ROS increased in blood and denervated muscle within 24 h, peaking at 12 h, mirroring neutrophil kinetics (n=4/group; P<0.05 or P<0.01 vs 0 h). - ROS inhibition with L-NAC significantly reduced neutrophil accumulation at 6 h and 12 h compared with saline controls (n=4/group; P<0.05, P<0.01), indicating ROS-dependent activation/recruitment. - Intravascular behavior and tissue infiltration: - SD-IVM visualized rolling, crawling, and accumulation of neutrophils within denervated muscle microvessels, peaking at 12 h; FITC-albumin showed patent vessels without leakage up to 14 days. - 3D reconstructions of serial sections confirmed neutrophils outside large and small vessels within denervated muscle tissue, demonstrating extravasation and tissue infiltration. - Neutrophil subsets and chemotaxis: - Two subsets identified in muscle: CXCR2+ (dominant) and CCR2+. CXCR2+ neutrophils peaked at 12 h and declined by day 3; CCR2+ cells peaked at day 1 and remained elevated at day 7 (n=4/group; P<0.05, P<0.01 vs 0 h). - RNA-seq at 6 h post-denervation: 678 DEGs (357 up, 321 down). Enriched GO terms included neutrophil chemotaxis and CXCR chemokine receptor binding; KEGG pathways included cytokine–cytokine receptor interaction and IL-17 signaling. - CXCL1 was among the top 10 upregulated genes; qPCR showed CXCL1 mRNA increased by 3 h and peaked at 12 h (n=4/group; P<0.05), supporting CXCL1–CXCR2 axis–mediated recruitment. - Functional role in atrophy: - Neutrophil depletion (DT in Ly6g-DTR-GFP mice; anti-Ly6G in WT) exacerbated denervation-induced gastrocnemius weight loss at 7 days versus saline/IgG controls (n=6/group; P<0.05). HE analyses showed smaller mean fiber area and diameter and higher fiber density at 7 days with depletion (P<0.05). No significant differences at 14 days. - Interpretation: Neutrophils mitigate early atrophy within the first week post-denervation. - Resolution via P53-mediated apoptosis: - P53 KO mice exhibited reduced muscle weight loss and larger mean fiber area at 1 week versus WT (n=6/group; P<0.05), paralleling protective neutrophil effects. - P53 KO had increased neutrophil numbers at 7 days versus WT, with no difference at 14 days (n=4/group; P<0.05). Neutrophil p53 levels increased and peaked at 12 h post-denervation. At 12 h, P53 KO mice showed fewer TUNEL-positive apoptotic neutrophils versus WT (n=4/group; P<0.05), indicating that neutrophils undergo P53-mediated apoptosis within 7 days, limiting their numbers.

The study demonstrates that denervation, despite lacking direct muscle injury, elicits a rapid, ROS-associated inflammatory response that mobilizes neutrophils to denervated skeletal muscle. CXCR2+ neutrophils, recruited via CXCL1, dominate early infiltration, extravasate into muscle tissue, and functionally delay early atrophy. This extends the paradigm of neutrophil involvement from direct injury sites to indirectly affected tissues and identifies ROS–CXCL1/CXCR2 signaling as the axis coordinating recruitment. The subsequent decline in neutrophil numbers is mediated by P53-dependent apoptosis, supporting a programmed resolution phase that restores homeostasis. These insights suggest that temporally tuned modulation of neutrophil activation/recruitment or survival could therapeutically mitigate early denervation-induced atrophy while preserving resolution mechanisms.

This work provides in vivo and 3D spatial evidence that ROS-activated, CXCL1-recruited CXCR2+ neutrophils infiltrate denervated skeletal muscle, where they delay early atrophy before undergoing P53-mediated apoptosis. It identifies key molecular nodes—ROS, CXCL1/CXCR2, and P53—governing neutrophil fate and function in denervated muscle. Future research should define the downstream effector mechanisms by which neutrophils protect muscle (e.g., debris clearance, trophic signaling, vascular remodeling), delineate contributions of CCR2+ subsets, optimize timing/dosing to enhance protective neutrophil actions without impairing resolution, and evaluate translational relevance in larger models and human denervation conditions.

- The precise mechanisms by which neutrophils delay atrophy were not elucidated; protective mediators and interactions with muscle or stromal cells remain undefined. - Functional outcomes beyond histology and wet weight (e.g., strength) were not reported. - The protective effect was characterized primarily within the first week; later time points showed no differences with depletion, limiting conclusions on longer-term roles. - Findings are based on mouse models; generalizability to human denervation requires validation.