Staphylococcus aureus (S. aureus) biofilm is the major cause of failure of implant infection treatment that results in heavy social and economic burden on individuals, families, and communities. Planktonic S. aureus attaches to medical implant surfaces where it proliferates and is wrapped by extracellular polymeric substances, forming a solid and complex biofilm. This provides a stable environment for bacterial growth, infection maintenance, and diffusion and protects the bacteria from antimicrobial agents and the immune system of the host. Macrophages are an important component of the innate immune system and resist pathogen invasion and infection through phagocytosis, antigen presentation, and cytokine secretion. The persistence, spread, or clearance of infection is determined by interplay between macrophages and S. aureus in the implant infection microenvironment. In this review, we discuss the interactions between S. aureus biofilm and macrophages, including the effects of biofilm-related bacteria on the macrophage immune response, roles of myeloid-derived suppressor cells during biofilm infection, regulation of immune cell metabolic patterns by the biofilm environment, and immune evasion strategies adopted by the biofilm against macrophages. Finally, we summarize the current methods that support macrophage-mediated removal of biofilms and emphasize the importance of considering multi-dimensions and factors related to implant-associated infection such as immunity, metabolism, the host, and the pathogen when developing new treatments.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

Staphylococcus aureus (S. aureus) is a common Gram-positive pathogen that causes a wide variety of diseases ranging from mild wound infections to fatal endocarditis, pneumonia, osteomyelitis, and sepsis [[1]–[4]]. Implant-associated infection caused by S. aureus is one of the main reasons for the failure of clinical treatment [5]. After invading the human body and colonizing the surface of the implant, S. aureus may switch from individual planktonic cells to clusters of structured bacteria wrapped in a cellular matrix, forming the notorious biofilm [6] that develops a powerful resistance to antibiotics and host immune responses.

As an important component of the innate immune system, macrophages are the first line of defense against pathogen invasion, and they are responsible for a wide range of immune activities, including phagocytosis of foreign bodies and microorganisms, antigen presentation, neutrophil recruitment, secretion of inflammatory cytokines (such as TNF-α, IL-1, and IL-6), and efferocytosis [7]. As one of the determinants of infection clearance or maintenance, the distinct immune phenotype of macrophages, formed by classical or alternative pathways, influences the outcome of infection [8].

The role of immune cells in S. aureus infection has been extensively discussed in several reviews [[9]–[11]]. However, the mechanisms of immune evasion against macrophages and advanced macrophage-associated biofilm eradication strategies used by the S. aureus biofilm have not yet been systematically discussed. In this review, we summarize the interaction between biofilms and macrophages during S. aureus infection. The complex relationship between immune cells and biofilms is discussed from the perspectives of the S. aureus biofilm structure, the effects of the biofilm on macrophage immune responses and immune metabolism, and the strategies used by the biofilm to evade macrophages. We also review current biofilm clearance strategies that modulate the interactions between the biofilm and macrophages and approaches that assist macrophages in disrupting biofilm formation or eradicating existing biofilm. Subsequently, we compare and contrast the advantages and disadvantages of each method and suggest research directions that may lead to the most effective strategies.

Biofilm Development and Component: The Origin of the Story

The process of biofilm formation has been demonstrated in vitro [[6], [12]–[14]] and can be divided into five general stages: single cell attachment, bacteria proliferation, exodus, biofilm maturation, and dispersal [15]. S. aureus attaches to the surface of implants through bacterial surface adhesins [16], wall teichoic acids [17], and eDNA [[18], [19]]. Hydrophobicity, electrostatic, and van der Waals force interactions are also involved in its attachment [20]. The adherent bacteria proliferate and produce an extracellular matrix (ECM) that allows bacteria to gather and form a complex and stable multilayered structure. The ECM is mainly composed of polysaccharides, proteins, teichoic acid, and eDNA, but the contents of each component vary among isolates. For example, methicillin-sensitive S. aureus strains use polysaccharides as the main biofilm component, whereas protein-dependent biofilm is more typical in methicillin-resistant S. aureus (MRSA) strains [[21], [22]]. The biofilm polysaccharide is known as a polysaccharide intercellular adhesin (PIA), which is also known as poly-β(1–6)-N-acetylglucosamine [23]. It has a net positive charge that facilitates aggregation and attachment. PIA/poly-β(1–6)-N-acetylglucosamine synthesis depends on the icaADBC operon [24]. Biofilms formed by MRSA strain-derived proteins tend to be ica operon-independent [25]. S. aureus surface proteins, including autolysins AtlA [26], fibronectin-binding factors A/B (FnBPA/B) [22], serine-aspartate repeat protein (SdrC) [27], S. aureus surface-associated protein G (SasG) [28], biofilm-associated protein (Bap) [29], and accumulation-associated protein (Aap) [30], promote biofilm matrix development and maintain its stability. In addition to these, eDNA exists as a conserved component in the biofilm of different bacteria genera. In vitro experiments have shown that biofilm formation is significantly increased in thermostable nuclease (nuc) mutant strains [31] but is decreased as a result of AtlA mutation [32], which is the main factor regulating the release of eDNA. Negatively charged eDNA can bind to PIA with a positive charge via electrostatic interactive forces, or it can non-covalently crosslink with cell wall-associated proteins to bridge the bacteria together [33].

Although polysaccharides, proteins, and eDNA are the main components of biofilm substrates, the contents of these biofilm components change dynamically in relation to different environmental factors and different growth stages. For example, S. epidermidis strains isolated from a high-shear stress environment produce more PIA-dependent biofilms than those isolated from low-shear environments [34], which suggests the importance of studying temporal and spatial factors when analyzing biofilm structure [35].

Macrophage Inflammatory Phenotype: Vital for Biofilm Clearance or Persistence

Due to the inflammatory milieu and differentially expressed genes, which result in the differential expression of cell surface markers and the secretion of specific cytokines, macrophages can be divided into classically activated pro-inflammatory and alternatively activated anti-inflammatory macrophages. Numerous studies have equated M1 and M2 as pro-inflammatory and anti-inflammatory macrophages, respectively. However, it is inappropriate to describe macrophages using M1/M2 nomenclature in the context of implant infection because the initial purpose of the M1/M2 dichotomy was to describe the phenotype of macrophages under tightly controlled in vitro conditions [36], whereas the situation in vivo is far more complex. In addition, several publications have reported that macrophages can simultaneously exhibit M1 and M2 gene expression profiles [36]. Orecchioni et al. [37] compared the transcriptome data of macrophages activated classically or alternately in vivo and in vitro and found that the gene expressions of macrophages that were stimulated in the same way exhibited significant differences in vitro and in vivo. Avital [38] infected primary human macrophages with various Gram-positive bacteria (including S. aureus) to investigate macrophage inflammatory dynamics. A single-cell transcriptome analysis revealed that the responding macrophages had multiple discrete cellular states, and these cellular states dynamically altered the inflammatory trajectory as the infection persisted; this reflected the heterogeneity and plasticity of macrophage transcription following the onset of infectious inflammation in vivo. Therefore, in this review, macrophages are distinguished based on their activation status and functional characteristics rather than being referred as M1/M2.

The debate regarding whether biofilm-derived S. aureus and phagocytosed planktonic bacteria remain within the macrophage phagolysosome or escape the vacuole is largely dependent on the conditions of the interaction and may still be a topic of discussion. In general, during planktonic bacterial infection, macrophages are activated through classical toll-like receptor 2 (TLR-2) and 9 (TLR-9) pathways to phagocytose bacteria, produce pro-inflammatory factors, such as iNOS, TNF-α, IL-1β, and IFN-γ, and generate reactive oxygen species (ROS) to enhance bacteria clearance [[10], [39], [40]]. TLR-2 on the macrophage surface recognizes various S. aureus-derived pathogen-associated molecular patterns (PAMPs), including lipoproteins, peptidoglycan (PGN), and lipoteichoic acid [[41]–[43]], and this triggers the TLR-2-dependent MYD88-related signaling pathway and inflammasome activation in phagosomes [[44], [45]]. Macrophages can also identify the unmethylated CpG motif characteristics of bacterial DNA via the intracellular receptor, TLR-9 [46]. Upon phagosome phagocytosis and digestion, bacterial DNA is released and binds to TLR-9, triggering NLRP3 inflammasome activation and oxidative stress and increasing superoxide anion production [47]. In the stage of S. aureus biofilm infection, biofilm can evade the recognition of classical receptors [48], thereby promoting bacterial survival within the host. Microenvironment and pathogenic virulence factors will skew macrophages toward the anti-inflammatory phenotype; upregulate arginase 1 (Arg-1), IL-4, and IL-10; downregulate iNOS; attenuate bactericidal activity; and promote fibrosis around the biofilm to prevent macrophage invasion and phagocytosis [[5], [49]]. When mice are infected with S. aureus biofilm, macrophages predominantly exhibit an anti-inflammatory phenotype and attenuate the pro-inflammatory response [50]. Increased anti-inflammatory macrophages were observed in a rat model with an S. aureus biofilm-associated prosthetic joint infection (PJI) [51]. Additionally, at the late stage of S. aureus infection, alveolar macrophages are more likely to exhibit an anti-inflammatory phenotype [52]. These reports suggested that the biofilm promotes anti-inflammatory polarization (Fig. 1).

Fig. 1.

Overview of macrophage interactions with planktonic S. aureus and biofilm. Different stages of S. aureus infection influence macrophage polarization and cytokine secretion toward pro-inflammatory or anti-inflammatory. Planktonic S. aureus can be recognized by TLRs and phagocytosed by macrophages, which destroy the bacteria through antimicrobial mechanisms, leading to pro-inflammatory responses. However, biofilm can evade these mechanisms and proliferate within macrophages, driving macrophages toward an anti-inflammatory phenotype, leading to bacterial persistence, host cell death, and chronic infection. This process is driven by the infection microenvironment and biofilm-derived signals that are largely unknown (indicated by question mark). Furthermore, MDSCs inhibit T cell and macrophage immunity, inducing immune tolerance and exacerbating the infection. PAMPs, pathogen-associated molecular patterns; TLR-2, toll-like receptor 2; TLR-9, toll-like receptor 9; iNOS, inducible nitric oxide synthase; Arg-1, arginase 1; ROS, reactive oxygen species; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; MDSCs, myeloid-derived suppressor cells; IL-4, interleukin-4; IL-10, interleukin-10; IL-12, interleukin-12; IL-17, interleukin-17; TGF-β, transforming growth factor-β.

The different phenotypic characteristics of macrophages and their inflammatory responses are critical for biofilm clearance. By adoptively transferring activated anti-inflammatory macrophages into mice infected with early biofilms, Hanke [53] found that pro-inflammatory factors were significantly increased and biofilm formation was attenuated. In addition, the 3D-printed scaffolds containing active macrophages and antibiotics designed by Aldrich [54] promoted the eradication of S. aureus biofilm. These studies indicate that enhancing the pro-inflammatory activity of macrophages is a novel therapeutic strategy for overcoming local biofilm immunosuppression.

Myeloid-Derived Suppressor Cells: An Accomplice to Biofilm Infection

Several studies [[39], [55]] have reported the association between S. aureus biofilm infection and bone myeloid-derived suppressor cells (MDSCs), which are a highly heterogeneous cell population composed of immature monocytes and granulocytes that have immunosuppressive characteristics [56]. In accordance with morphological disparity, MDSCs can be divided into two distinct subsets, monocyte-like MDSC (M-MDSC) (CD11b+ CD14+CD33+HLA−DRlow/neg for human and CD11b+Ly6C+ for mouse) and polymorphonuclear MDSC (CD11b+ CD15+HLA−DRlowCD66b+ for human and CD11b+Ly6G+ Ly6Clow for mouse); the latter was also previously known as granulocyte-like MDSC (G-MDSC) [57]. Using scRNA-seq, Aldrich analyzed the abundance and trajectory of immune cells (including MDSCs, macrophages, and neutrophils) in a mouse model of S. aureus cranial infection. MDSCs were a prominent infiltrate in the infected microenvironment, and they outnumbered PMN in the galea and bone flap; this emphasizes their crucial role in infection [58].

Heim et al. [59] identified MDSCs as a key factor in chronic infection caused by S. aureus biofilm, and they demonstrated that MDSCs negatively modulate host immune responses in S. aureus-associated PJI by suppressing T cell activation and limiting secretions of T cell-related cytokines, including TNF-α, IFN-γ, and IL-17. Inhibition of T cell functions by MDSCs was related to arginine metabolism [60]. Extracellular arginine depletion results in the decreased expression of the CD3ξ chain [61] and failure to upregulate cyclin D3 and CDK4, thereby leading to low T cell reactivity and cell cycle arrest [[62], [63]]. Despite the increased Arg-1 expression and arginine consumption at the site of S. aureus biofilm infection, it was found that myeloid-derived Arg-1 does not critically affect orthopedic implant-associated and catheter-associated S. aureus biofilm infection, but it serves a distinctive role in planktonic infection [64]. This further confirms the different immune responses to planktonic and biofilm infections. The proportion of CD4+ CD25+Foxp3+Tregs in CD4+ lymphocytes was greatly increased after the co-culture of spleen T cells and MDSCs treated with S. aureus biofilm, and it was speculated that MDSCs may abrogate host immune response by stimulating Treg cell proliferation [51]. TGF-β and IL-10 play crucial roles in Treg development [[65], [66]]. Both G-MDSC and M-MDSC increase levels of TGF-β and IL-10, and the effect of M-MDSC is more pronounced than that of G-MDSC [67]. Therefore, it is believed that MDSCs can promote the proliferation of Tregs by upregulating TGF-β and IL-10 during S. aureus-related PJI, thus inhibiting inflammatory reactions at the infected sites of prosthetic joints and culminating in chronic infection.

Peng et al. [51] demonstrated that M-MDSCs, rather than G-MDSCs, could be converted into macrophages with anti-inflammatory properties when stimulated by S. aureus biofilm in vitro and in vivo. However, the infiltrating MDSCs in PJI in vivo were found to be predominantly granulocytic [56], and this was confirmed in clinical samples from patients with PJI rather than those with aseptic loosening, which suggests the feasibility of using G-MDSCs as markers of infection to assist in the differential diagnosis of PJI [55]. Heim et al. [[68], [69]] infected a mouse PJI model with S. aureus biofilm and showed that the recruitment of MDSCs was associated with IL-12 from infected tissues and that the anti-inflammatory properties of MDSCs were achieved through the release of IL-10 to regulate the activation of anti-inflammatory macrophages. As IL-10 is one of the major inducers of anti-inflammatory macrophages, this finding may partially explain the transformation mechanism of the inflammatory macrophage phenotype. However, Tebartz et al. [70] showed that immunosuppression of MDSCs was not mediated by IL-10 or TGF-β, but it required direct cell-cell contact or proximity. With the loss of IL-12 or IL-10, the number of MDSCs decreased, the biofilm burden was reduced, and the pro-inflammatory activity of biofilm-associated monocytes was increased. The ability of macrophages to promote biofilm clearance in the absence of MDSCs was indirectly demonstrated. These results imply that biofilm-recruited MDSCs may regulate macrophage anti-inflammatory polarization and promote biofilm persistence [59]. In brief, MDSCs have immunosuppressive effects on S. aureus biofilm-associated PJI, but the detailed mechanisms remain unclear and require further analysis.

Macrophage-Related Immune Evasion: Essential Components for Biofilm Persistence

S. aureus biofilm generates diverse mechanisms that impair host immunity and evade the immune response. In this section, we focus on advances made in determining the macrophage avoidance mechanisms of S. aureus immune evasion, with the aim of providing novel insights for treatments of S. aureus biofilm infection (Table 1).

Table 1.

Macrophage-related immune evasion mechanisms

MechanismEvasion factorFunctionReferenceImmune recognition escapeSSL3, SSL4Block TLR-2 activation[73, 74]CoagulaseBinds fibronectin to EPS, shields PAMPs[75]Capsular polysaccharideBlocks PAMP recognition, protects against opsonophagocytosis[76]Tolerance to phagocytosis and macrophage invasionHla, LukABInhibit macrophage phagocytosis via agr-dependent pathway[79]FnBPAInhibits macrophage invasion and aggravates biofilm development[80]Promotion of anti-inflammatory activityDacASynthesizes c-DI-AMP, promotes S. aureus survival in macrophages, and enhances anti-inflammatory activity[81]Virulence factorsInterfere with PRR-dependent NF-κB pathway[82–84]Biofilm-derived lactateInhibits HDAC11, promotes IL-10 transcription[86]AtpAPromotes biofilm formation, produces toxins and proteases, and regulates MDSCs and macrophage infiltration and inflammation[90]Escape from Immune Recognition

S. aureus can evade recognition by macrophage surface receptor TLRs. Staphylococcal superantigen-like (SSL) proteins, a family of exoproteins with similar superantigen structures but without superantigenic activity, play an essential role in this process [[71]–[73]]. Among the 14 members of the SSL family (SSL1-14), the immune evasion mechanism of SSL3 was first elucidated. SSL3 can block TLR-2 activation by direct extracellular interaction, inhibiting recognition of heat-inactivated S. aureus, PGN, or lipoprotein TLR-2 ligands and downregulating TNF-α in murine macrophages [[74], [75]]. The removal of sialic acid residues reduces the binding of SSL3 to TLR-2, which suggests that the SSL3-TLR-2 interaction is partially dependent on the sugar chain [74]. Of the other SSL family members, only SSL4 can block TLR-2, and this is consistent with the high homology between SSL4 and SSL3. In addition to the direct interactions described above, S. aureus biofilm can construct mechanical barriers that shield PAMPs on the surfaces of bacteria to achieve immune evasion. For instance, to protect bacteria from immune recognition, S. aureus can bind fibronectin to its extracellular polymeric substances by expressing coagulase [76]. Capsular polysaccharide also prevents macrophages from recognizing PAMPs, masks complements from detection, protects against opsonophagocytosis, and decreases TNF-α of mouse macrophages [77].

Tolerance to Phagocytosis and Macrophage Invasion

Biofilm-derived S. epidermidis exhibit better intracellular survival and persistence in macrophages after phagocytosis and induce lower production of pro-inflammatory cytokines and Th1-responsive cytokines, such as TNF-α, IL-12p40, IL-12p70, and IFN-γ, compared with planktonic bacteria [[78], [79]]. Similarly, the expressions of IL-1β, TNF-α, CXCL2, and CCL2 were found to be significantly reduced during S. aureus biofilm infection in mice. In addition, macrophage invasion into the biofilm was limited, the microbicidal immune phenotype was deviated, iNOS expression was decreased, and strong Arg-1 induction was observed. When co-cultured with S. aureus biofilm, macrophages displayed limited phagocytosis and a gene expression pattern similar to that of alternately activated anti-inflammatory macrophages [50]. This may be related to the synergistic effect of α-toxin and leucocidin AB on the inhibition of phagocytosis and cytotoxicity [80]. Contributions of α-toxin and leucocidin AB to macrophage dysfunction and biofilm development partly depend on the accessory gene regulator [80], which is the quorum-sensing system of S. aureus that controls the production of most toxins and enzymes. FnBPA inhibits macrophage invasion and aggravates biofilm development in a SaeRS two-component system-dependent manner [81].

Promote Anti-Inflammatory Activity

Biofilms can disrupt the anti-inflammatory and pro-inflammatory balance of macrophages in the microenvironment, thereby upregulating anti-inflammatory responses and contributing to infection maintenance. DacA, the enzyme responsible for c-DI-AMP synthesis, is highly expressed during the growth of biofilm. The c-DI-AMP is released into ECM via bacterial cell lysis, which induces the expression of macrophage type I IFN by a STING-dependent pathway, promoting S. aureus survival in macrophages and enhancing anti-inflammatory activity [82]. Various virulence factors secreted from S. aureus biofilm can interfere with the pattern recognition receptor-dependent NF-κB pathway, thereby inhibiting the antimicrobial and pro-inflammatory responses of host cells [[83], [84]]. Alboslemy et al. [85] co-cultured a biofilm-conditioned medium with mouse macrophages and attenuated activation of pro-inflammatory transcription factor NF-κB, whereas the expression of NF-κB negative regulator, Kruppel-like factor 2 (KLF2), was upregulated. The authors hypothesized that KLF2 in host cells can act as a target of S. aureus virulence factors during biofilm infection, which aggravates anti-inflammatory responses and destroys the innate immune reactions of macrophages. This is also supported by Nakayama et al. [86], who found that S. aureus interacted with paired immunoreceptor tyrosine-based inhibitory motif-containing Ig-like receptors on murine macrophages to inhibit NF-κB activation, thereby negatively interfering with TLR-mediated cytokine production. Another study [87] showed that biofilm-derived lactate can inhibit histone deacetylase (HDAC11) in host cells, which results in the acetylation of histone 3 in IL-10 promoter, and promotes IL-10 transcription in MDSCs and macrophages. The increased expression of IL-10 then inhibits T cell activation and Th1 polarization [[88], [89]], thus tilting macrophages toward an anti-inflammatory phenotype [90].

Bosch et al. [91] screened the Nebraska Transposon Mutant Library and found that AtpA, the operon encoding the alpha subunit of ATP synthase of S. aureus, is vital for biofilm formation, toxin and protease production, and regulating MDSCs and macrophage infiltration and inflammation. AtpA mutation leads to the formation of a diffuse biofilm structure that facilitates leukocyte infiltration, and toxin and protease secretion are also reduced. In addition, pro-inflammatory cytokines IL-12p70, TNF-α, and IL-6 from macrophages are significantly increased, giving rise to biofilm clearance.

Immune Metabolism: A New Perspective on Biofilm Infection

A growing amount of evidence indicates that the anti-inflammatory/pro-inflammatory phenotype of macrophages is controlled by metabolism [[8], [92], [93]]. As mentioned above, the activation status of macrophages affects biofilm clearance. Understanding and elaborating the functions of immune cells in fighting infection from the perspective of metabolism, and exploring the role of macrophage-related metabolic mechanisms in innate immunity, have gradually become innovative directions in the field of infection and immunity [[94]–[96]] (Fig. 2).

Fig. 2.

S. aureus infection affects macrophage metabolism to regulate the inflammatory response. Macrophages respond to planktonic infections via TLRs, triggering the expression of markers such as iNOS, CD80, CD86, and MHC-II and the release of inflammatory factors, such as IL-1β, TNF-α, IFN-γ, IL-6, and IL-12. Glycolysis-based metabolic processes provide anabolic intermediates for the pro-inflammatory response, and the fatty acid synthesis and pentose phosphate pathway are also involved, which are associated with the disruption of the TCA and formation of ROS. In contrast, biofilm infection polarizes macrophages to an anti-inflammatory state. The cellular metabolism is dominated by oxidative phosphorylation (OxPhos) and fatty acid oxidation (FAO), which may involve several receptors associated with anti-inflammatory cytokines (i.e., CD36, IL-10R, IL-4R, IL-13R, A2R) as well as transcription factors (i.e., STAT3, STAT6, PPARγ), which leads to the production of cytokines, such as Arg-1, TGF-β, IL-10, IL-4, IL-13, IL-8, and VEGFA. Nutrient and oxygen gradients in the tissue microenvironment also influence the macrophage inflammatory response phenotype, which is closely related to the metabolic state of macrophages. Given the distinct immune metabolism modes of macrophages during planktonic and biofilm infections, a promising therapeutic approach for biofilm infection would be to regulate immune function by interfering with key nodes of the macrophage metabolic pathway, which have been marked in red (glycolysis, OxPhos, and IDH and SDH in Krebs cycle). G-6-P, glucose-6-phosphate; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; OxPhos, oxidative phosphorylation; TCA, tricarboxylic acid cycle; HIF-1α, hypoxia inducible factor-1α; IDH, isocitrate dehydrogenase; SDH, succinate dehydrogenase; Acetyl-CoA, acetyl-coenzyme A; α-KG, alpha-ketoglutarate; u-PFK2, ubiquitous 6-phosphofructo-2-kinase/fructose bisphosphatase-2; iNOS, inducible nitric oxide synthase; NO, nitric oxide; Arg-1, arginase 1; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; MHC-II, major histocompatibility complex class 2; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6; PPARγ, peroxisome proliferator-activated receptor-γ; FAO, fatty acid oxidation; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-12, interleukin-12; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; TGF-β, transforming growth factor-β; VEGFA, vascular endothelial growth factor A; IL-10, interleukin-10; IL-4, interleukin-4; IL-13, interleukin-13; IL-8, interleukin-8; IL-4R, interleukin-4 receptor; IL-10R, interleukin-10 receptor; A2R, adenosine receptor.

The main feature of pro-inflammatory macrophages is to increase carbon flux and promote the production of ROS, NO, and other antimicrobial substances through aerobic glycolysis [97]. Stimulated by pro-inflammatory signals, such as the pattern recognition receptor recognition of pathogens, the pentose phosphate pathway in macrophages is activated, u-PFK2 (a highly active phosphofructokinase isoform) is upregulated, and key nodes of the TCA cycle are disrupted, thus, causing the intracellular accumulation of citrate, succinate [98], and itaconic acid [99]. The metabolic changes described above contribute to pro-inflammatory cytokine secretion, inflammasome activation [100], pro-inflammatory lipid biosynthesis, and enhancement of the superoxide burst effect of the NADPH oxidase complex [101], which ultimately maintain macrophage inflammatory responses.

In contrast, anti-inflammatory activities in macrophages are driven by mitochondrial oxidative phosphorylation, and fatty acid oxidative metabolism is also involved [102]. Processes that strengthen glycolysis in pro-inflammatory macrophages are downregulated in anti-inflammatory macrophages. For example, iNOS is lowly expressed in anti-inflammatory macrophages, whereas Arg-1 is highly expressed and preferentially metabolized to urea and ornithine [103]. The latter is further metabolized to polyamines and proline, which may lead to granuloma and fibrosis, causing tissue remodeling, infection restriction, and abscesses [104]. To facilitate triglyceride uptake, strengthen the TCA cycle, and reduce glycolysis, anti-inflammatory macrophages express PFKB1 (a less active PFK2 isoform compared to u-PFK2) and upregulated CD36 [105].

Gradients of oxygen, nutrients, metabolite, and proton concentrations in the biofilm-infected microenvironment can reprogram macrophage metabolism and alter their inflammatory profile [[106], [107]]. The formation of gradients is the result of competition and interactions between the biofilm and host cells. However, this process becomes more complicated if multiple pathogens inhabit the biofilm. Although most bacteria in S. aureus biofilm are dormant and have a lower metabolic activity than those with a planktonic status, the abundance of bacteria may allow the biofilm to consume oxygen and nutrients continuously, thereby creating a microenvironment devoid of oxygen and nutrients, reducing macrophage aerobic glycolysis, and facilitating anti-inflammatory polarization [108]. Meanwhile, the anaerobic milieu can induce expressions of biofilm-associated virulence genes, such as icaADBC, which encodes PIA and promotes bacterial aggregation and adhesion [[23], [109]]. The mechanisms involved in this phenotypic change are currently unknown, but Yarwood et al. [110] compared the homology of the ResDE two-component system of Bacillus subtilis with that of S. aureus, and they initially established the role of the SrrAB two-component system in controlling the switch between aerobic and anaerobic respiration modes in S. aureus. Ulrich’s team [108] further confirmed that SrrAB is a major activator of ica expression and PIA production in anaerobic environments and that it helps protect S. aureus from non-oxidative defense mechanisms. However, the respiratory burst of host cells in the early stage of planktonic infection consumes large amounts of oxygen [[111], [112]], which is also the cause of nutrient depletion in the local environment. A lack of blood supply in the implant environment, especially in bone tissue, may further exacerbate the anaerobic and nutrient-deficient environment and the anti-inflammatory polarization of immune cells.

Taken together, these findings show that the metabolic properties of macrophages can affect the immune response to bacteria and interactions with other immune cells. S. aureus does not only undergo its own metabolic reprogramming during infection, but it also affects the metabolic capacity of the host. The pathogen-host and host-host interactions increase complexity involved in conducting cellular metabolism studies. Considerable caution must be taken when conducting relevant in vitro studies, as mammalian cell culture media components may deviate substantially from in vivo metabolite concentrations, which may result in considerable differences between the actual metabolic changes seen in vivo infections and those obtained in vitro [113].

Treatment Strategy: Targeting Macrophages

S. aureus biofilm is characterized by high antibiotic and host immune resistance provided by the ECM barrier, which is rich in polysaccharides, proteins, and eDNA. S. aureus remains dormant in the anoxic environment; its metabolic activity is low, and it is insensitive to external stimuli, which further maintains biofilm persistence. The current options assisting biofilm clearance by macrophages can be broadly classified into the following categories: (a) use of antimicrobial agent combinations using new drug delivery systems; (b) application of physical, chemical, or enzymatic methods to disperse biofilms and expose the bacteria to antibiotics and the immune system; (c) methods targeting the immune system and key nodes of immune-related pathways to enhance host immune responses; (d) and those modifying implant materials to modulate macrophage phenotypes and hinder S. aureus attachment and biofilm development (Fig. 3).

Fig. 3.

Strategies to facilitate macrophage-mediated S. aureus biofilm clearance. Biofilm can be eradicated by using a combination of antimicrobial agents and new drug delivery systems, such as exosomes, liposomes, and nanoparticles, which can penetrate into the biofilm and disrupt it. Physical and chemical methods, biological enzymes, and proteins have also been applied to disperse biofilm. Improving host responses via targeting the immune system and key nodes of immune-related pathways is also a useful biofilm treatment strategy. Modifying implant materials to protect implants from S. aureus attachment and to modulate the phenotype of macrophages is a promising way to prevent biofilm formation. MΦ, macrophage; US-PCCA, ultrasound-stimulated phase-change contrast agent; LSW, laser shockwave; US, ultrasound; PDT, photodynamic therapy; d-AA, d-amino acid.

Antibacterial Agent Combination and Delivery

The use of prolonged administration of antibiotics at high doses is the traditional method employed to treat biofilm infections [114]. However, high concentrations of antibiotics can be cytotoxic and may lead to the development of antibiotic-resistant strains. S. aureus survives in macrophage phagocytosis [115], and it can protect itself from antibiotics via low intracellular retention or poor intracellular accumulation [116], which makes host cells a source of bacteria propagation. Thus, there is an urgent need to develop novel drug-targeted delivery systems, nonantibiotic antimicrobials, and antibiotic adjuvants that enable biofilm clearance.

Exosomes are nanoscale, bilayer membrane vesicles secreted by cells [117]. Due to their low immunogenicity, high blood stability, and target specificity, exosomes are ideal natural drug carriers [118]. Yang et al. [119] used mannose-modified exosomes as drug carriers in targeted drug delivery to macrophages with a high level of mannose receptors and targeted drug transport to bacterial infection sites via macrophage recruitment. In addition, the antibiotic concentration was reduced by using a combination of vancomycin and lysostaphin; this decreased cytotoxicity and drug resistance, thereby providing a new method of assisting macrophages in eradicating intracellular bacteria. Similarly, liposomes and polymeric nanoparticles are currently being investigated as potential drug delivery vehicles due to their strong ability to penetrate the biofilm matrix [[120]–[122]].

Persisters in biofilm have a low metabolic activity and are tolerant to most antibiotics [123]. In addition, biofilm can act as a physical barrier that impedes the diffusion of antibiotics and the invasion of macrophages [124]. Combining persister-targeted antibiotics with a biofilm bacteria membrane penetration enhancer is a useful strategy for addressing these issues. Previous studies [[123], [125]] have shown that rhamnolipids produced by P. aeruginosa can induce the uptake of aminoglycosides by Gram-positive bacteria, and the barrier can be disrupted using an ultrasound-stimulated phase-change contrast agent to improve drug penetration [124]. The combination of rhamnolipids and aminoglycosides with ultrasound-stimulated phase-change contrast agent significantly improves the ability to destroy MRSA biofilm [124]. This new strategy has the potential for rapid clinical translation, as PCCA is already employed in clinical practice [124]. Meanwhile, the use of bacteria membrane penetration enhancers and antibiotic adjuvants may suggest another feasible idea for assisting macrophages in biofilm clearance.

Biofilm Dispersion

Another method used to destroy biofilm is to disrupt and disperse its natural barrier and tight structure, respectively, using physical, chemical methods and enzymes that restore the bacteria to a planktonic state and increase their susceptibility to antibiotics and the immune system. The current physical methods employed include laser shockwave, ultrasound, and photodynamic therapy (PDT) [[126], [127]]. Laser shockwave employs a high-energy shockwave and ultrasound uses a sound wave frequency that is above the human hearing range: both release energy that disperses the biofilm. PDT irradiates biofilm using specific light wavelengths that activate photosensitive drugs gathered in the biofilm; this triggers a photochemical reaction to produce ROS that destroy bacteria [128]. Combining PDT with antibacterial drugs synergistically provides another method of effectively treating biofilm-associated infections. Willis [129] performed PDT with photosensitizer methylene blue in combination with chloramphenicol and tetracycline to reduce the resistance of MRSA. Xiu [130] further tested the effect of PDT on modulating the physicochemical conditions of the biofilm infection microenvironment. PDT potentiated the hypoxic microenvironment, induced the anaerobic metabolism of MRSA, and activated the antimicrobial activity of metronidazole. In addition, PDT-activated chemotherapy polarized macrophages to an anti-inflammatory phenotype and promoted the repair of biofilm-infected wounds; this suggests that the immunomodulatory effects of PDT on immune cells have a potential value.

Chemical methods comprise the use of various types of plant extracts and plant-derived compounds, d-amino acids, and fatty acid signaling molecules. Essential oils and plant extracts can inhibit and disperse S. aureus biofilm [131] by disrupting bacterial quorum-sensing systems [132], and they have also been used together with antibiotics. The natural compound cuminaldehyde was used synergistically in combination with ciprofloxacin against S. aureus biofilms [133]. In addition, the effect of d-amino acids in biofilm dispersion was identified in Bacillus subtilis, where the binding of d-amino acids to PGN caused the failure of matrix protein TasA to attach to the cell wall and a reduction in intercellular adhesion [134]. Subsequently, Hochbaum [135] observed similar phenotypes in S. aureus and P. aeruginosa, and it was also suggested that d-amino acids could inhibit biofilm-associated protein accumulation. A further study confirmed that d-amino acids could disrupt eDNA [136]. Fatty acid signaling molecules can restore dormant cells within the biofilm to a metabolically active state, promoting bacterial diffusion [137]. Harrison [138] synthesized 2-heptylcyclopropane-1-carboxylic acid, an analog of cis-2-decanoic acid, which locks the signaling factor in an active state and prevents isomerizing to inactive trans-configuration, thereby significantly improving biofilm dispersion. Combining this with antibiotics reduced the minimum antibiotic concentration required for biofilm inhibition and eradication.

Bioenzymes can promote biofilm decomposition by efficiently degrading the biofilm matrix or the S. aureus cell wall. Bacterial cell wall hydrolases, such as lysostaphin [139] and phage-derived endotoxin [140], degrade pentaglycine bridges in S. aureus cell walls, and disrupt bacterial aggregation. S. aureus-derived proteases, such as V8 protease [141], metalloproteinase, aureolysin (Aur) [142], and cysteine protease (staphopain A) [143], can break down biofilm and expose bacteria to immune cells. Exogenous proteases, including proteinase K [144] and trypsin [145], have also been found to reduce ECM stability. Different sources of nucleases, such as staphylococcal nucleases [31] and recombinant human DNase (DnaseI) [146], have antibiofilm capabilities, but they have no obvious effect on mature biofilms. Finally, glycogen hydrolases, such as α-amylase [147], dispersant B [148], and hyaluronidase [149], can degrade polysaccharides in ECM, thereby reducing interbacterial aggregation and surface adhesion.

Yu et al. [150] exogenously provided the protein PslG with endoglycosidase characteristics, which disrupted the Psl exopolysaccharide matrix to disperse P. aeruginosa biofilms, enhanced biofilm susceptibility to antibiotics and macrophages, and improved biofilm clearance. Although the effect of PsIG was pathogen dependent, this result demonstrated the potential synergistic effect of biofilm dispersants on host immune bactericidal function. It is therefore suggested that to better model the complex environment in vivo, subsequent studies should focus on the interaction between pathogen, host, and biofilm dispersion factors.

As described above, the natural barrier structure of S. aureus biofilm enables it to circumvent the traditional TLR-2 and TLR-9 bacterial recognition pathways, attenuate the host pro-inflammatory response, and limit macrophage phagocytosis [50]. We reviewed several methods that effectively disrupt biofilm, increase the susceptibility of bacteria to immune cells, and enable indirect biofilm clearance. However, the vast majority of studies focus on the effects of biofilm dispersion, whereas limited studies have simultaneously examined the effects of macrophage-dependent bactericidal actions. As it is difficult for macrophages to penetrate S. aureus biofilm, and the small fraction of penetrated macrophages are functionally attenuated in this niche [50], we suggest that a more feasible biofilm clearance approach would be to disrupt the biofilm structure so that macrophages are enabled to clear the infection.

Immune Response Improvement

There are some promising methods used to remove biofilm: targeting key nodes with immune-related pathways, modulating immune cell profiles (particularly those of macrophages with an inflammatory phenotype), and boosting the host immune response. Yamada [151] significantly reduced the S. aureus biofilm burden from the perspective of immunometabolism. By targeting monocytes with nanoparticles carrying oligomycin (an oxidative phosphorylation inhibitor), they successfully reprogrammed the metabolism and improved the pro-inflammatory properties of infiltrating monocytes. Systematically co-administered antibiotics were applied on this basis, which effectively cleared the PJI biofilm infection in mice. To treat MRSA catheter-associated infected mice, Hanke [53] targeted macrophages with the C5a receptor (C5aR/CD88) agonist EP67 to induce the production of pro-inflammatory mediators by CD88+ MΦs. In addition, Guo [152] proposed a spatially selective chemodynamic therapy strategy: CuFe5O8 nanocubes were used as catalysts to reverse the immunosuppressive microenvironment by generating −OH to induce pro-inflammatory macrophage polarization. Pro-inflammatory immune responses and −OH eliminated biofilm debris and continuously exposed the bacteria.

MRSA is resistant to several pro-inflammatory agents, including antimicrobial peptides and NO−, which enables it to evade inflammatory clearance and results in skin and soft tissue infections [[153], [154]]. The host response must eventually transition to a resolution phase, during which anti-inflammatory macrophages with a high Arg-1 expression generate MRSA-toxic spermine and spermidine via spermidine metabolism [153]. Given that proliferator-activated receptor γ (PPARγ) is essential for anti-inflammatory macrophage polarization and intracellular polyamine production [[155], [156]], Thurlow et al. [157] tested the necessity of bone marrow-specific PPARγ for the transition of the immune response from an inflammatory state to a resolution state. The requirement of PPARγ for abscess formation was also demonstrated. The application of PPARγ agonists accelerated the transition from an inflammatory to a resolution phase, leading to accelerated abscess formation and the enhanced clearance of S. aureus. This beneficial effect was achieved mainly through innate immune cells, especially macrophages. These results once again prove that the immunomodulatory antimicrobial method is a potential avenue that can be followed to develop treatments that combat the growing threat of antibiotic-resistant microbes.

Modifications of Implants

The implant surface is the preferred biofilm infection site. There is an acute demand for the development of new implant materials and the modification of material surface properties that hinder S. aureus adhesion and biofilm formation. Changes in the kinetic factors of surface materials, such as hydrophobicity [158] and flatness [159], can effectively reduce S. aureus adhesion and aggregation. Antimicrobial-containing coatings have also been applied to control implant infections [160]. Material coatings can modulate the macrophage phenotype and are thus indirectly antimicrobial. Nanofilms formed by ZnO nanoparticles immobilized on titanium surfaces can prohibit sessile bacteria and enhance the antibiofilm efficacy of macrophages and PMNs by promoting phagocytosis and the secretion of inflammatory cytokines [161].

Advantages and Disadvantages of Current Treatment Strategies

S. aureus biofilm formation, the immune evasion mechanisms of biofilm, and strategies formulated to combat these problems have been well reviewed and extensively discussed [[9], [35], [162]–[165]]. In this section, we review advanced research that focuses on assisting macrophages to interfere with biofilm formation or eradicating existing biofilm, and we summarize several strategies employed to combat S. aureus biofilm infections.

Antibiotics are the most commonly used antibiofilm method in clinical practice. However, cytotoxicity and tissue damage caused by excessive concentrations of antibiotics are concerning side effects. The spread of drug-resistant bacteria caused by the misuse of antibiotics is also a threat to public health. Although many studies have focused on the development of novel drug-targeted delivery systems, nonantibiotic antimicrobial agents, and antibiotic adjuvants, the effectiveness of their practical application and effect on host immunity in vivo is still unknown.

A variety of approaches targeting biofilm barriers have been investigated, and promising biofilm lysis results have been seen in vitro; however, few studies have integrated these approaches with host cells or the immune system. Bacteria are released after the dispersion of biofilm, which risks the spread of infection if they are not treated in combination with an effective bactericidal therapy.

Using immune cells as anti-infective targets, the selective pressure for antimicrobial resistance to evolve can be largely avoided. Furthermore, due to the nonspecific characteristics of innate immune defenses, immunomodulation provides broad-spectrum protection against a wide range of microbial pathogens. However, over-activated innate immunity can result in deleterious pro-inflammatory responses and tissue damage. Therefore, we suggest that further research targeting immunomodulatory therapies should focus on how to stimulate protective immunity in a controlled manner without increasing the systemic pro-inflammatory response.

High doses of antibiotics are often systemically administered to reach implant-associated biofilm infection sites and clear bacteria, and this results in toxicity. Implants are platforms for the adherence of S. aureus and biofilm formation. By releasing a drug locally through material coatings to increase the concentration of the drug within the region of infection, improved therapeutic effects can be obtained at lower drug dosages, and the risk of liver and kidney damage caused by high doses of antibiotics can be avoided. However, antimicrobial approaches used in material coatings that are based on the release of substances run the risk of depletion, and the efficacy of surface-related mechanisms may diminish due to coverage by proteins adsorbed from body fluids. Therefore, the choice of the implant coating method employed should depend on the clinical application target and whether the expected effect is short or long term.

The microenvironment of the implant infection is characterized by complex interactions between the host, the implant, and the pathogenic bacteria. We therefore suggest that the combined use of multiple anti-infection strategies may be the optimum future direction. In this respect, we suggest that studies should focus on modifying implant surface properties to inhibit S. aureus adhesion, enhancing host immune cells to fight infection, and developing novel antimicrobial agents to promote biofilm dispersion and bactericidal activity.

Conclusion and Future Perspective

The S. aureus biofilm, a complex biome, is formed by a community of planktonic bacteria that colonize implant surfaces and proliferate on them. Macrophages are important members of the immune system that defend against pathogen invasion and destroy foreign bodies. Uncovering the complex interaction between the biofilm and macrophages will provide a clearer understanding of the relationship between infection and immunity, and this will assist in the development of effective antimicrobial tools. Early in the invasion of S. aureus, planktonic bacteria stimulate macrophage activation; this causes a pro-inflammatory response and phagocytosis, which destroys bacteria. However, as the infection persists, macrophages shift to an anti-inflammatory phenotype. The S. aureus biofilm further increases the difficulty of clearing the bacteria by evading macrophage recognition of PAMP, tolerating phagocytosis, reprogramming host cell metabolic patterns, promoting anti-inflammatory polarization, and inducing cell death. With the progression of research focusing on biofilm and the infection microenvironment, new treatment strategies are being developed. However, most strategies have been based on the results of in vitro or in vivo studies in mice, and data on safety and efficacy in humans and actual clinical situations are currently lacking. The generation of different clinical isolates and drug-resistant mutant strains are also major challenges for providing effective clinical treatment. Given the complexity of the in vivo environment, the differences in the inflammatory properties of immune cells in response to planktonic and biofilm infections, and the interplay between immunity and metabolism, it is suggested that future studies should integrate host and pathogenic factors to determine the most appropriate therapeutic pathway.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This review was funded by the Natural Science Foundation of China (Grant No. 82272511 and No. 82202727), Shanghai Science and Technology Commission (Grant No. 21140904800), and China Postdoctoral Science Foundation (Grant No. 2022M712109).

Author Contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work. L.M. and Y.J. contributed equally to this work. Y.J. conducted the literature search and planned the review. L.M. wrote the first draft of the manuscript. G.G. provided technical suggestions. S.H. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

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