Complement in chronic wound infections: complementary or a cascade of chaos?

Introduction

Chronic wounds are defined as wounds that fail to heal after 3 months of treatment. In Australia, each year it is estimated that 450,000 people suffer from chronic wounds, costing the health care system well over A$6.6 billion.¹ These wounds also have a profound negative effect on the patient’s quality of life and psychosocial wellbeing. Underlying conditions such as venous insufficiency, atherosclerosis, and type-2 diabetes influence the development of non-healing wounds.² These wounds are often colonised by numerous bacterial species, which complicate healing, delay wound closure and negatively affect health outcomes.³ The precise relationship between the wound microbiome, biofilm production and dysregulated immune response in chronic wounds remains poorly understood.⁴

Normal v. chronic wound healing

Wound healing is a complex physiological process that occurs over four main phases including haemostasis, inflammation, proliferation and remodelling.⁵ Acute wounds typically progress through these phases in an orderly and timely manner, as illustrated in Fig. 1a.^6,7 The early inflammatory responses are important to drive initial wound healing, remove foreign debris and prevent infection. By contrast, chronic wound healing often stalls somewhere in the late inflammatory phase before wound closure can occur. This leads to hypergranulation at the wound edges, decreased fibroblast function, excessive and persistent inflammation, and ultimately immune dysfunction.^5,7–9 These pathological changes contribute to fibrosis and the development of non-healing ulcers, impairing proliferation and preventing tissue repair (Fig. 1b).

Fig. 1.

Acute v. chronic wound healing with host–pathogen interactions. (a) Typical progression of acute wound healing is depicted through the four main phases. (b) By contrast, chronic wound healing stalls in the late inflammatory stage. (c) A comparison of the immune responses and microbial clearance mechanisms in acute (left) and chronic (right) wounds. Acute wounds exhibit adequate inflammation and cell infiltration facilitating pathogen clearance utilising neutrophil extracellular traps (NETs) and phagocytosis. Additionally, commensal skin bacteria such as Staphylococcus may inhabit the wound. Chronic wounds, however, are often infected with biofilm-forming Gram-negative bacteria such as P. aeruginosa, which penetrate deeper tissue layers. These wounds are characterised by excessive inflammation, immune dysregulation, and increased antimicrobial resistance. Created in BioRender (see https://BioRender.com/0wu586v).

Host–pathogen interactions and immune dysregulation in chronic wounds

Bacteria in wounds can significantly affect both local and systemic immune responses; however, the relationship between bacterial burden and the extent of these immune alterations are unclear.⁴ Following skin injury, the innate immune system is activated in acute wounds, leading to the recruitment of neutrophils and macrophages, and the induction of pro-inflammatory cytokines including Intereukin-6 (IL-6), Interleukin 1β (IL-1β), and tumour necrosis factor (TNF).⁷ Antimicrobial peptides (AMPs) are also produced and include α- and β-defensins, cathelicidins and histatins^8,10 (Fig. 1c left). Acute wound colonisation by Staphylococcus aureus is common and increases immune cell recruitment compared to aseptic wounds.¹¹

By contrast, in chronic wounds there is often increased bacterial abundance and diversity, particularly of Gram-negative species.⁴ Pseudomonas aeruginosa is a common wound pathogen that can impair wound healing by dysregulation of immune responses, such as suppression of vascular endothelial growth factor (VEGF) and S100A8/A9 AMP production, with increased neutrophil infiltration.^3,4 In the chronic wound stage, some AMPs such as human β-defensins hBD2 and hBD3 can be further upregulated.^4,8 In fact, Histadin-1-3 and P113 have progressed to clinical trials that target chronic P. aeruginosa wound infections.¹⁰

Bacterial biofilms refer to polymicrobial aggregates that are embedded within a protective matrix of extracellular polymeric substances and are present in up to 90% of chronic wounds.³ Biofilms produced by Gram-negative bacteria can release significant levels of endotoxins into the wound environment, promoting antimicrobial resistance, impairing tissue repair, and perpetuating inflammation^4,8 (Fig. 1c right). Quorum sensing within biofilms facilitates bacterial communication and upregulation of virulence factors, enhancing the bacteria’s ability to trigger an inflammatory response and delay wound healing.³ Persistent inflammation may further contribute to the dysfunctional cellular and pro-inflammatory cytokine responses characteristic of chronic wounds. These include elevated numbers of inflammatory cells (including neutrophils, macrophages and Langerhans cells), excessive production of Interleukin 1α and TNF, as well as defects in phagocytosis and apoptosis, and impaired phenotypic switching of pro-inflammatory macrophages (M1) to an anti-inflammatory phenotype (M2)^5,8,9 (Fig. 1c right). Biofilm mechanisms aside, polymicrobial infections with pathogenic organisms are important for chronic wound formation.⁴ The use of probiotics in wounds can potentially assist in the reduction of microbial load by species antagonism and immunomodulatory mechanisms; however, the heterogeneity of results thus far limits this as a viable treatment option.¹²

Complement in wound healing and bacterial interactions

The complement cascade is a key component of the innate immune system, consisting of a proteolytic cascade involving up to 50 molecules that defend against invading pathogens. This system is activated by three pathways that converge at the cleavage of complement component C3, cleaving C3 into C3a and C3b. C3b subsequently initiates cleavage of C5 into C5a and C5b. C5b in combination with several other complement proteins forms the membrane attack complex (MAC), which creates pores in the bacterial membranes, leading to pathogen lysis.¹³ C3a and C5a are important anaphylatoxins and drive inflammatory responses for vasodilation and inflammatory cell infiltration, with C5a being the most potent.^14,15 Complement is produced systemically and locally in the tissues, where it contributes to infection control and wound healing.¹⁴ However, as with all inflammatory processes, balance is essential. Whereas complement helps control infection, dysregulation can perpetuate tissue damage.

C5a is implicated in the pathogenesis of many inflammatory diseases and some infectious diseases.¹⁵ In a study of pneumonia patients, elevated serum C5a was associated with increased inflammatory IL-6 and C reactive protein compared to healthy controls. Additionally, Pneumococcus-infected mice treated with anti-C5a antibodies experienced less severe disease.¹⁶ Similarly, C5a receptor knock-out (KO) mice infected with Escherichia coli in a pyelonephritis model had a lower bacterial load and reduced renal lesions when compared to wild-type (WT) mice.¹⁷ It appears that targeting C5a or its receptor may lead to reduced tissue damage. Elevated levels of C5a were detected in wound fluid of a diabetic murine model compared to WT controls, therefore modulation of C5a may also protect against excessive wound damage.¹⁸

Mice lacking C3 or C5 showed reduced inflammation, neutrophil infiltration and increased rate of healing 48 h post-wounding. Only the C3 KO group had increased mast cells, which they hypothesised led to increased vascularity and accelerated healing.¹⁹ Conversely, topical application of C3 and C5 in a collagen carrier in rats was associated with improved wound healing and strength compared to collagen carrier alone.^14,20 These contrasting findings may reflect the differences between local and systemic complement activity. For instance, in a porcine burn wound model, prolonged elevation of serum C3 levels were observed up to 60days post-injury, likely leading to complement dysregulation, while local C3 levels in the wound remained unchanged.²¹ Importantly, complement activity may differ between animal species so direct comparison should be avoided. Few studies have investigated complement in human chronic wounds, with one reporting elevated serum C3 in patients with chronic leg ulcers.²²

Knowledge of bacterial–complement interactions in wounds is also lacking. Cystic fibrosis lung P. aeruginosa isolates were reported to produce proteases (AprA and LasB) that degrade C5a, helping modulate host immune responses. These proteases were more active in acute isolates, suggesting that reduced C5a-cleavage by chronic isolates may contribute to complement dysregulation in persistent infections, including in P. aeruginosa-infected chronic wounds.²³ Elastase-producing P. aeruginosa strains were shown to partially degrade C3 and components of wound fluid when introduced into plasma.²⁴ In another study, enzymatic treatment targeting P. aeruginosa exopolysaccharides (Pel and Psl), critical for biofilm formation, increased C3 deposition in pooled serum, and attenuated wound infection in a murine model.²⁵ These studies demonstrate that P. aeruginosa can target C3, likely interfering with complement effector functions such as MAC, to inhibit bacterial clearance and wound healing. Yet overall, the role of complement dysregulation in chronic wound infections, particularly in relation bacterial interactions with C3, C5 and their split products, remains poorly understood and should be a key focus of future research.

Future perspectives on pathogen–complement interaction research

Studying complement–bacterial interactions presents significant challenges due to the complexity and instability of the complement system. Complement proteins are heat labile and inherently unstable, making the appropriate selection and handling of specimens crucially important. Serum is generally preferred for functional complement analysis, whereas EDTA plasma is more suitable for stabilisation of complement split products, due to the chelation of calcium and magnesium to inhibit further complement activity.²⁶ Citrate and heparin plasma are considered less suitable due to inadequate chelating ability, and negative complement component interactions respectively^26,27 (Fig. 2). Despite these general recommendations, sample-specific validation is important. For example, one study found similar C5a levels across serum, EDTA plasma and citrate plasma, but noted increased C4d and C3a in serum. If study constraints limit access to multiple sample types, properly handled serum may serve as a versatile alternative for broader complement studies.

Fig. 2.

Recommended workflow for collection, storage and analysis of specimens for wound complement research. This schematic outlines optimal steps for studying complement activity in wounds with considerations of specimen type, timing of collection and storage, and analysis methods for functional studies or detection of individual components. Created in BioRender (see https://BioRender.com/7mmt8yc).

Optimal preservation of complement activity requires rapid freezing at −80°C with minimal freeze–thaws cycles, ideally on ice,²⁷ yet this may be unpractical in a clinical or research setting. Complement biomarkers in citrate and EDTA plasma can stay stable within 24 and 48 h respectively when stored promptly at 4°C.²⁸ Prolonged storage beyond these timeframes, however, has been associated with increased levels of C4d and C3a. Less is known about the optimal handling of complement-containing wound fluids. Nevertheless, C5a has been successfully detected from filter paper secured in place within the wound for various timepoints.¹⁸ In theory, fluid collected during wound irrigation or debridement with saline could be used for complement analysis if processed in the presence of protease inhibitors to stabilise complement components.

Irrespective of sample type, enzyme-linked immunosorbent assays (ELISA) remains the most reliable method for quantifying individual complement proteins and cleaved products.^26,27 In the context of bacterial infection, complement deposition can be assessed qualitatively using immunofluorescent staining of tissue sections or qualitatively by flow cytometry with gating for bacterial cells.^21,29 Additionally, serum bactericidal assays are a common method for assessing bacterial sensitivity to complement and are widely used in our laboratory.³⁰ An optimised workflow to guide complement sample handling and testing is shown in Fig. 2. For a more detailed guide describing complement activation assessment methods, readers are referred to the following review.²⁷

Conclusion

This article highlights how the persistent inflammation in chronic wounds predisposes tissues to ongoing infection and perpetuates a cycle of inflammation and impaired healing. Although the microbial contribution to chronic wound pathology remains a ‘chicken-and-egg’ scenario, current evidence suggests a positive feedback loop exists between bacterial persistence and immune dysregulation. The role of complement dysregulation in wounds deserves further investigation, particularly in relation to localised wound responses. To advance our understanding, future complement-focused research should prioritise rigorous sampling protocols, appropriate specimen processing and storage, and robust laboratory analyses.