Wound healing is a complex and delicately balanced biological process that normally progresses through distinct yet overlapping phases: hemostasis, inflammation, proliferation, and remodeling.
When functioning optimally, this intricate orchestra of cellular responses and molecular signals restores tissue integrity and function.
However, disruptions in any phase—particularly prolonged inflammation—can derail the entire healing trajectory, leading to chronic, non-healing wounds that cause significant patient suffering, economic burden, and potential long-term complications including amputation.
At the center of many chronic wound pathologies lies a paradoxical player: the neutrophil.
While these immune cells are essential first responders that help protect wounds from infection, their persistent presence and unregulated activity can transform them from guardians to saboteurs in the healing process.
Role of Neutrophils in Normal Wound Healing
Neutrophils are the most abundant white blood cells in circulation and serve as the body’s first line of cellular defense against invading pathogens. In the context of wound healing, neutrophils are rapidly recruited to the injury site, typically arriving within hours after tissue damage 5.
Clinical observations support their importance, as neutropenic individuals (those with abnormally low neutrophil counts) often experience difficulty with wound healing, highlighting the essential role these cells play in the initial repair process 5.
Upon reaching the wound site, neutrophils perform several critical functions. As professional phagocytes, they engulf and eliminate potential pathogens while clearing cellular debris from damaged tissues 5. They release antimicrobial substances including reactive oxygen species (ROS), proteases, and antimicrobial peptides that create a hostile environment for microorganisms 2.
Neutrophils also form neutrophil extracellular traps (NETs) – web-like structures composed of DNA, histones, and antimicrobial proteins that can capture and neutralize pathogens 8.
Beyond their antimicrobial functions, neutrophils actively shape the inflammatory response through the production of cytokines and growth factors that activate other cells important for the repair process 2.
They contain over 700 proteins stored in their segmented nucleus and granules that can be rapidly released upon activation, contributing directly to regeneration and revascularization independent of transcription 5.
In normal wound healing, neutrophil activity is tightly regulated.
After performing their protective functions, neutrophils undergo apoptosis (programmed cell death) and are subsequently cleared by macrophages 2 5.
This clearance process initiates a pro-resolution program characterized by the release of tissue-repairing cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) 5.
The uptake of apoptotic neutrophils by macrophages serves as a critical signal for the resolution of inflammation, allowing the wound to progress through subsequent healing phases 2.
Neutrophil Persistence in Chronic Wounds
While a robust neutrophil response is essential in acute wounds, the persistence of active neutrophils is a hallmark of chronic, non-healing wounds 2. In these cases, what begins as a protective inflammatory response fails to resolve, creating a self-perpetuating cycle of inflammation and tissue damage.
Multiple factors can trigger this transition from acute to chronic inflammation, including venous hypertension, diabetes, prolonged tissue pressure, and impaired blood supply leading to tissue hypoxia 6.
In chronic wounds, inflammatory cells continue to be recruited and activated despite the absence of ongoing infection or injury 2.
This persistent inflammation prevents the wound from progressing through the proper stages of repair.
High levels of neutrophil-derived proteases, such as serine proteases and matrix metalloproteinases (MMPs), have been consistently reported in chronic, non-healing wounds 2.
Excessive neutrophil infiltration appears to be a critical factor in this cycle of chronic inflammation and acts as a biomarker of chronic wounds 3. The persistent presence of neutrophils at the wound site delays the healing process through continued expression of pro-inflammatory factors, proteases, and NETs 3.
This results in additional tissue destruction that causes persistent inflammation and more tissue damage, preventing progression through the proper stages of repair 2.
Molecular Mechanisms
The weapons neutrophils deploy to fight infection can cause significant collateral damage when unleashed in an unregulated manner. Several key mechanisms contribute to neutrophil-mediated tissue injury in chronic wounds:
Protease Overproduction
Neutrophils release various proteases, including elastase, cathepsin G, proteinase 3, and MMPs, which under normal conditions help degrade damaged tissue components and kill pathogens 2. However, in chronic wounds, excessive protease production becomes destructive.
These enzymes can cleave growth factors and growth factor receptors, rendering them inactive and disrupting signals necessary for healing 2.
Additionally, they degrade the extracellular matrix (ECM) to such an extent that it becomes an ineffective substrate for cellular migration, creating a hostile environment for repair and enlarging the area in need of healing 2 6.
Oxidative Damage
While ROS production by neutrophils is crucial for antimicrobial defense, excessive ROS generation in chronic wounds leads to oxidative damage of the wound, impairment of neovascularization, and metabolic disruption3.
This oxidative stress can cause significant damage to the ECM and cell membranes, resulting in poor angiogenesis and re-epithelialization, insufficient production of growth factors, and prolonged inflammatory response 3.
In diabetic wounds, advanced glycation end products (AGEs) can induce excessive ROS production, exacerbating oxidative damage and aging of the ECM and cell membranes 3.
The combination of low nitric oxide (NO) content and excessive ROS production creates an environment hostile to proper healing, contributing to the chronicity of wounds 3.
Double-Edged Neutrophil Extracellular Traps
NET formation represents another double-edged sword in the wound healing process. While NETs help capture and eliminate pathogens during acute inflammation, excessive or dysregulated NET production can impede wound healing 8.
NETs are increased in diabetic foot ulcer (DFU) patients compared to healthy individuals, and components of NETs such as citrullinated histone 3 have been suggested as potential negative markers for wound healing 3.
Excessive NET formation may amplify inflammation and hinder wound healing through several mechanisms, including affecting wound structures, cellular functions, and angiogenesis8. NETs can also sustain inflammatory responses by activating other immune cells and prolonging the inflammatory phase.
Studies have shown that deletion of key NETosis enzymes or inhibition of the “NLRP3 inflammasome-NETs” inflammatory loop can improve angiogenesis and accelerate wound healing 6.
The Cellular Ecosystem in Chronic Wounds
Wound healing is orchestrated by complex interactions among various cell types, and neutrophils play a central role in this cellular ecosystem. In chronic wounds, dysfunctional neutrophil interactions with other cells propagate inflammation and impede healing:
Disrupted Macrophage Polarization
Under normal conditions, macrophages phagocytose apoptotic neutrophils, a process termed efferocytosis, which triggers the transition from pro-inflammatory M1 to pro-resolving M2 macrophage phenotypes 2 5.
In chronic wounds, this process is disrupted. Despite the increased presence of M1 macrophages, their ability to clear apoptotic neutrophils is inhibited6.
This leads to neutrophil accumulation and prevents the macrophage phenotypic shift necessary for resolution of inflammation and progression to tissue repair.
The dysregulated neutrophil-macrophage interaction creates a vicious cycle where inflammation persists and amplifies. Neutrophil recruitment promotes the production of more NETs and activates the release of NLRP3 inflammasomes and IL-1β from macrophages through the TLR-4/TLR-9/NF-κB signaling pathway 6.
This perpetuates the pro-inflammatory state, preventing the transition to the proliferative and remodeling phases of wound healing.
Impact on Fibroblasts and Keratinocytes
The prolonged inflammatory environment in chronic wounds profoundly affects stromal cells. Fibroblasts exposed to this inflammatory milieu upregulate inflammatory gene expression rather than focusing on ECM production and remodeling6.
Meanwhile, keratinocytes release IL-1α and type I interferon, triggering an inflammatory chain reaction in adjacent stromal cells and promoting further immune cell recruitment 6.
The large number of pro-inflammatory factors increases the release of MMPs, accelerating ECM degradation and reducing fibroblast proliferation and collagen deposition 6. This compromises the structural integrity of the healing tissue and delays or prevents wound closure.
T Cell Involvement
The sustained inflammatory response in chronic wounds leads to activation and accumulation of T cells, especially Th17 cells 6. These cells secrete IL-17 to maintain M1 macrophage activity, leading to persistent inflammatory wound healing 6. This represents another layer of immune dysregulation that contributes to the chronicity of inflammation and impaired healing.
Neutrophil Trafficking and Fate
Recent research has revealed more complex neutrophil behaviors than previously recognized, including various forms of neutrophil trafficking, death mechanisms, and even the ability to leave inflammatory sites.
Neutrophil Heterogeneity and Migration
Emerging evidence suggests that neutrophils exhibit considerable heterogeneity and can adopt various fates in inflamed tissues 7.
Beyond the traditional view of neutrophils as short-lived cells that either die at inflammatory sites or are cleared by macrophages, research now indicates more complex behaviors, including swarming around infectious or damaged sites and various forms of cell death (apoptosis, necroptosis, NETosis) 7.
Particularly interesting is the phenomenon of neutrophil swarming, which has been observed in various models of sterile inflammation and infection 7.
While swarming can limit tissue damage and infection in normal conditions, excessive or uncontrolled swarms may contribute to aggravation and propagation of tissue damage, as seen in pulmonary inflammation and inflammatory flares in gout disease7.
Neutrophil Reverse Migration
An alternative neutrophil fate has recently gained attention: reverse migration, where neutrophils move away from inflammatory sites back into the vasculature 1 4. This process may play dual roles in inflammation resolution or dissemination.
Recent research has identified molecular mechanisms involved in this process, including the interaction between C-C motif chemokine receptor-like 2 (CCRL2) on neutrophils and its ligand chemerin in plasma 1.
Using sophisticated tracking methods in an air pouch model, researchers demonstrated that neutrophils undergoing reverse migration show increased CCRL2 expression, while chemerin concentrations in plasma increase during the late inflammatory stage 1.
Neutralizing chemerin decreased the proportion of reverse-migrating neutrophils in blood, suggesting that circulating chemerin attracts neutrophils to leave inflammatory sites by interacting with CCRL21.
This mechanism may contribute to either the resolution of localized inflammation or, conversely, the dissemination of inflammation to distant sites 1 4.
The statement that “some studies have reported that neutrophils would leave the site of damaged or infected tissue in a process termed neutrophil reverse migration” indicates this is an emerging area of research with potentially significant implications for understanding and treating chronic wounds4.
Altered Neutrophil Death Mechanisms
In normal wound healing, neutrophil apoptosis followed by macrophage clearance is essential for resolving inflammation 2 5. However, in pathological conditions, neutrophils may undergo alternative forms of cell death that can exacerbate tissue damage and inflammation.
Necroptosis, a form of programmed necrotic cell death, has been observed in neutrophils under certain inflammatory conditions7.
For example, phagocytosis of methicillin-resistant Staphylococcus aureus can induce necroptosis in human neutrophils, allowing the escape of viable bacteria, liberation of intracellular neutrophil contents, and generation of IL-1β, which aggravate local tissue injury and lead to persistent infection7.
The activation of necroptotic pathways (such as RIPK3-MLKL) has been detected in neutrophils from inflamed tissue samples from patients with neutrophilic diseases, suggesting that neutrophil necroptosis occurs under in vivo inflammatory conditions 7.
This form of inflammatory cell death may contribute to the perpetuation of inflammation in chronic wounds.
Targeting Neutrophils in Non-Healing Wounds
The central role of neutrophils in chronic wound pathology suggests several therapeutic strategies to promote healing:
Modulating Neutrophil Activity
Rather than completely suppressing neutrophil function, which could compromise antimicrobial defense, therapies could aim to modulate neutrophil activity to limit tissue damage while preserving protective functions.
This might include selective inhibition of specific neutrophil-derived proteases or targeting ROS production without eliminating essential antimicrobial capacity.
Promoting Resolution of Inflammation
Drugs that promote neutrophil apoptosis have therapeutic potential to accelerate tissue repair 5. By enhancing the normal resolution processes, such approaches could help chronic wounds transition from the inflammatory to the proliferative phase.
Research has shown that promoting neutrophil apoptosis provides a strong signal for the resolution of inflammation, allowing the wound to continue through subsequent healing phases 2.
NET-Targeting Approaches
Given the detrimental effects of excessive NET formation in chronic wounds, therapies targeting NETs are showing promise 8. Anti-NET therapies have exhibited effectiveness in improving wound healing, as demonstrated in several studies 8.
These approaches might include inhibiting NET formation enzymes, degrading existing NETs, or blocking their downstream inflammatory effects.
Neutrophil Reverse Migration Modulation
The discovery of neutrophil reverse migration mechanisms offers potential new therapeutic targets. Manipulating the CCRL2-chemerin axis might provide a way to regulate neutrophil egress from inflamed tissues1.
However, more research is needed to determine whether promoting reverse migration would help resolve localized inflammation or risk spreading inflammatory processes to distant sites.
Conclusion
The transformation of neutrophils from essential defenders to destructive agents in chronic wounds exemplifies the double-edged nature of inflammation in tissue repair.
While neutrophils are crucial for initial wound decontamination and setting the stage for healing, their persistent presence and unregulated activity can sabotage the very repair processes they are meant to support.
The pathology of chronic wounds involves multiple interdependent mechanisms, including excessive neutrophil infiltration, overproduction of proteases and ROS, formation of cytotoxic NETs, disrupted apoptosis and clearance, and dysregulated interactions with other immune and stromal cells.
These processes create a self-perpetuating cycle of inflammation that prevents progression to the proliferative and remodeling phases of wound healing.
Recent discoveries regarding neutrophil heterogeneity, alternative death mechanisms, and the phenomenon of reverse migration have expanded our understanding of these cells’ roles in wound healing and chronicity.
These insights open new avenues for therapeutic intervention that go beyond simply suppressing neutrophil recruitment or activity.
The challenge for future wound care approaches lies in balancing the beneficial antimicrobial functions of neutrophils with their potential for tissue damage.
By targeting specific aspects of neutrophil biology—such as excessive protease activity, NET formation, or altered migration patterns—we may develop more nuanced strategies to promote resolution of inflammation and facilitate healing in chronic wounds.
As research continues to unravel the complex biology of neutrophils in wound healing, we can anticipate more precise and effective interventions for the millions of patients suffering from non-healing wounds worldwide.
The goal remains to harness the protective potential of these remarkable immune cells while preventing their transformation into enemies of the healing process.
References
- https://www.nature.com/articles/s41419-024-06820-5
- https://pmc.ncbi.nlm.nih.gov/articles/PMC3763227/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC9104327/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC10363828/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC5820392/
- https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.789274/full
- https://journals.physiology.org/doi/full/10.1152/ajpcell.00181.2020
- https://www.nature.com/articles/s41419-021-04294-3