Reprogramming Macrophage Phenotypes With Photobiomodulation for Improved Inflammation Control in ENT Organ Tissues

Ken Woo; Yeon Soo Kim; Celine Abueva; Seung Hoon Woo

doi:10.21053/ceo.2024.00286

Journal List > Clin Exp Otorhinolaryngol > v.18(1) > 1516090458

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Woo, Kim, Abueva, and Woo: Reprogramming Macrophage Phenotypes With Photobiomodulation for Improved Inflammation Control in ENT Organ Tissues

Review Article

Clin Exp Otorhinolaryngol 2025;18(1):1-13.

Published online: 19 December 2024

DOI: https://doi.org/10.21053/ceo.2024.00286

Reprogramming Macrophage Phenotypes With Photobiomodulation for Improved Inflammation Control in ENT Organ Tissues

Ken Woo^1,^*

, Yeon Soo Kim^2,^*

, Celine Abueva³

, Seung Hoon Woo⁴

¹Department of Ecology and Evolutionary Biology, University of California, Los Angeles, Los Angeles, CA, USA

²Department of Otorhinolaryngology-Head and Neck Surgery, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Korea

³Beckman Laser Institute, Cheonan, Korea

⁴Department of Otorhinolaryngology-Head and Neck Surgery, Dankook University College of Medicine, Cheonan, Korea

Corresponding author: Seung Hoon Woo Department of Otorhinolaryngology-Head and Neck Surgery, Dankook University Hospital, 201 Manghyang-ro, Dongnam-gu, Cheonan 31116, Korea Tel: +82-55-750-8173, Fax: +82-55-759-0613 Email: lesaby@hanmail.net

^* These authors contributed equally to this article as co-first authors.

Received 21 September 2024 Revised 11 December 2024 Accepted 18 December 2024

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Photobiomodulation (PBM), a noninvasive phototherapy that utilizes wavelengths between red and near-infrared light, has emerged as a promising approach for controlling inflammation by modulating macrophage polarization. This review investigates the therapeutic potential of PBM in treating ENT-specific inflammatory conditions, such as chronic rhinosinusitis and otitis media, focusing on its effects on macrophage phenotypes and evidence from preclinical studies. By promoting mitochondrial activity, increasing adenosine triphosphate production, and modulating reactive oxygen species, PBM has been shown to shift macrophages from a pro-inflammatory to an anti-inflammatory phenotype. Studies have demonstrated that PBM enhances tissue repair, reduces inflammatory markers, and promotes wound healing. Moreover, PBM facilitates the polarization of M2 macrophages, a crucial factor in resolving mucosal inflammation in the nasal, pharyngeal, and middle ear cavities, as well as restoring tissue homeostasis. The anti-inflammatory effects of PBM are attributed to its ability to influence several molecular mechanisms involved in inflammation regulation, particularly in ENT organ tissues, where recurrent inflammation can lead to chronic conditions such as otitis media or sinusitis. Furthermore, this review compares PBM to competing methods for reprogramming macrophages and treating inflammation, highlighting its advantages of minimal toxicity, simplicity, and precision in controlling ENT immune responses.

Keywords: Photobiomodulation Therapy, Macrophage Inflammatory Proteins, Macrophage Activation, Inflammation, Otolaryngology

INTRODUCTION

When the body encounters pathogens, toxic compounds, irradiation, or harmful stimuli, the immune system mounts an inflammatory response, a key defense mechanism. Inflammation, an essential component of the innate immune system, includes chemical and physical barriers as well as cellular defenses. This is particularly important in ENT tissues, which are often exposed to pathogens and allergens [1]. While acute inflammation typically resolves infections or injuries and restores tissue homeostasis through a series of chemical signals and cellular interactions, uncontrolled inflammation in ENT tissues can lead to chronic conditions. These include chronic rhinosinusitis, otitis media, and laryngitis, all of which are characterized by swelling, redness, pain, and loss of tissue function [2-4].

Macrophages, which are key immune effector cells, play a significant role in inflammation and homeostasis by rapidly responding to stimuli and causing physiological changes [5]. These cells can differentiate into various subtypes, most notably the M1 (proinflammatory) and M2 (anti-inflammatory) phenotypes. In ENT organ tissues, such as the nasal passages and middle ear, M1 macrophages fight pathogens by releasing pro-inflammatory mediators. In contrast, M2 macrophages facilitate tissue repair and anti-inflammatory responses, thereby helping to restore mucosal integrity [6]. Although controlling macrophage polarization to adjust the M1/M2 balance is desirable for addressing harmful stimuli, the complexity of the involved signaling pathways presents significant challenges in standardizing these techniques [7].

Photobiomodulation (PBM) exerts noninvasive, nonthermal effects on ENT organ tissues, with two primary mechanistic hypotheses proposed: absorption by mitochondrial cytochrome c oxidase (CCO) and generation of reactive oxygen species (ROS). The CCO hypothesis posits that light in the 600–1,000 nm range stimulates mitochondrial respiration, thereby increasing adenosine triphosphate (ATP) production and enhancing anti-inflammatory responses, particularly through M2 macrophage polarization. Conversely, ROS generation through PBM may initially activate pro-inflammatory (M1) macrophages via oxidative stress. However, it could potentially shift toward anti-inflammatory (M2) phenotypes as ROS levels deplete and downstream pathways become activated [8,9]. Further research is necessary to determine the precise role of ROS in PBM-induced macrophage polarization. Although the exact mechanisms by which PBM affects cellular functions are not fully understood, studies indicate that it can promote cell viability, proliferation, differentiation, and migration [10].

Recent findings indicate the potential of PBM in modulating immune responses, particularly in the area of macrophage polarization, suggesting its clinical application for controlling inflammation. Therefore, considering the demonstrated benefits of PBM, it is essential to provide an overview of its role in stimulating inflammatory responses and influencing macrophage polarization to advance its therapeutic applications. This review summarizes the current understanding of the mechanisms behind inflammation and macrophage polarization and compares PBM with other polarization techniques, highlighting PBM’s potential to control inflammation caused by external stimuli.

MECHANISMS OF INFLAMMATION

The inflammatory response in ENT organ tissues involves a complex interplay of inducers, sensors, mediators, and effectors, forming intricate regulatory networks. This response is particularly notable when reacting to common irritants like allergens and pathogens in the nasal cavity and middle ear. Inducers, such as viral or bacterial infections in these tissues, initiate the inflammatory response by stimulating the production of mediators. These mediators then influence tissue states and facilitate adaptation to the inducers of inflammation. Subsequently, the mediators can propagate the inflammatory signal, recruiting various immune cells or promoting tissue repair processes [11].

Inducers of inflammation

Inducers of inflammation can be classified as either exogenous or endogenous. Exogenous inducers consist of microbial virulence factors and pathogen-associated molecular patterns (PAMPs). Virulence factors are specific microbial inducers that are not recognized by dedicated receptors and are exclusive to pathogens. These factors provoke inflammatory responses by damaging tissues. For instance, Gram-positive bacteria release pore-forming exotoxins that the NALP3 inflammasome detects through the resulting K⁺ ion efflux [12]. Conversely, PAMPs are molecular patterns found in microorganisms, whether commensal or pathogenic, and are directly recognized by the host’s pattern-recognition receptors (PRRs), which then initiate inflammatory responses [13].

Endogenous inducers arise from tissues that are stressed or malfunctioning and include signals such as ATP, uric acid, K⁺ ions, and S100 proteins, which are released during necrotic cell death. For instance, ATP released during necrotic cell death can bind to purinoreceptors on macrophages, triggering K⁺ ion efflux and activating the NALP3 inflammasome [14,15]. Similarly, the S100 calcium-binding protein family interacts with the receptor for advanced glycation end products and collaborates with Toll-like receptors (TLRs) to orchestrate an inflammatory response [16]. However, despite being classified based on reported tissue anomalies, the characteristics of these inducers remain poorly defined [11].

Mediators of inflammation

Inflammatory mediators, which include tissue-residing macrophages, mast cells, and local tissue cells, circulate in the plasma as inactive precursors [17]. During acute inflammation, these mediators become activated and their levels increase at specific sites. They can be categorized into seven groups: cytokines, chemokines, proteolytic enzymes, lipid mediators, vasoactive amines, and vasoactive peptides [18]. These mediators coordinate the delivery of blood components to sites of infection, enabling selective neutrophil extravasation while preventing the escape of erythrocytes [19]. When activated by pathogens or inflammatory cytokines, neutrophils release harmful substances such as ROS and reactive nitrogen species, contributing to the acute inflammatory response [5]. If this response is successful, macrophages shift to producing anti-inflammatory mediators, which support the resolution phase and promote tissue repair [20].

Key signaling pathways in inflammation

The regulation of the inflammatory response, which involves the combined effects of various immune cells, is governed by several key intracellular signaling pathways. These include the TLR, nuclear factor-kappa B (NF-κB), Janus kinase (JAK)/signal transducers and activators of transcription (STAT), and mitogen-activated protein kinase (MAPK) pathways. Fig. 1 concisely shows the significant role each of these pathways plays in initiating and maintaining the inflammatory response. This orchestration ensures an effective defense against pathogens while also preventing chronic inflammation.

TLR signaling

Among PRRs, TLRs are the most extensively studied and play a direct role in initiating inflammatory responses [21]. The TLR signaling cascade begins when PAMPs are recognized by PRRs on immune cells. Following PAMP binding, TLRs may activate the myeloid differentiation primary response 88 (MyD88) adaptor protein, which then triggers an intracellular signaling cascade. This cascade activates transcription factors such as activator protein-1 (AP-1), NF-κB, and interferon (IFN) regulatory factor 3 (IRF3). Together, these factors stimulate the expression of inflammatory cytokines and other mediators [22,23]. Conversely, TLRs can also initiate the Toll/interleukin (IL)-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)-dependent signaling pathway. In this pathway, TRIF engages tumor necrosis factor (TNF)- associated factor (TRAF) family members, such as TRAF3, initiating a series of phosphorylation reactions. These reactions lead to the formation of IRF3 dimers that move into the nucleus and promote inflammation [24-26].

NF-κB pathway

NF-κB activity is primarily induced by inflammatory cytokines and substances derived from pathogens. NF-κB, a transcription factor, plays a crucial role in the inflammatory process and consists of a family of proteins including p50, RelA (p65), and inhibitor of NF-κB (IκB) [27-29]. The activation of the NF-κB pathway can be initiated by various stimuli, such as inflammatory cytokines, leading to the recruitment of inflammatory cells. When activated, the IκB kinase (IKK) complex, comprising IKKα, IKKβ, and IKKγ, phosphorylates IκB proteins [30]. This phosphorylation triggers the degradation of IκB, allowing NF-κB to translocate into the nucleus and activate gene transcription, thereby promoting inflammatory responses [31]. This cascade results in the recruitment of immune cells and the production of pro-inflammatory cytokines, which contribute to the inflammatory response.

JAK/STAT pathway

The JAK/STAT pathway is a crucial signaling mechanism that includes cytokines, IFNs, growth factors, and hormones. It plays a pivotal role in translating extracellular signals into inflammatory gene expression [32]. This pathway features receptor-associated JAKs and STATs. When ligands such as cytokines bind to receptors, the associated JAK family proteins (JAK1, JAK2, JAK3, and TYK2) phosphorylate each other. This phosphorylation creates docking sites for STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) in the cytoplasm, which function as transcription factors [33]. These STAT proteins are subsequently phosphorylated, dimerize, and move to the nucleus. There, they bind to the promoter regions of target genes and actively regulate the expression of genes involved in inflammation [34]. During this signal transduction process, various regulators, such as protein inhibitors of activated STAT (PIAS) and protein tyrosine phosphatases, help maintain homeostasis with the inflammatory cytokines produced [35].

MAPK pathway

MAPKs are serine/threonine protein kinases that respond to various stimuli, including inflammatory cytokines, osmotic stress, and mitogens. They play crucial roles in regulating cell survival, differentiation, proliferation, and apoptosis [36,37]. In mammals, the MAPK family comprises extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases. Each MAPK pathway features a kinase cascade that includes a MAPK kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK [33]. For instance, the Raf protein kinase activates MKK1 and MKK2, with the signal subsequently reaching ERK1/2 in response to mitogens [38]. An alternative pathway involves MEKKs (a type of MAPKKK), such as MEKK1/4, ASKI, and MLK3, which activate MKK4 and MKK7. The downstream cascade then activates JNK and produces specific cytokines [39]. Another cascade features a different class of MEKKs, including MLK3, TAK, and DLK, which activate MKK3 and MKK6, leading to p38 activation in response to inflammatory stimuli. These activated MAPKs can lead to the phosphorylation and activation of transcription factors in the cytoplasm or nucleus, thereby initiating the inflammatory response [36,40].

MACROPHAGE POLARIZATION

Overview of macrophage subtypes

In addition to regulatory pathways, specific production of essential mediators by recruited immune cells, such as macrophages, is necessary for inflammatory responses to occur and be regulated in response to external stimuli. Macrophages are key players in ENT-related inflammation, adapting to environmental cues like allergens or infections in the nasal, pharyngeal, and middle ear mucosa by transitioning between different phenotypes [41]. M0 macrophages are undifferentiated macrophages that can polarize into their respective subtypes to perform specific functions in the context of inflammation [42]. Fig. 2 details the tightly controlled signaling pathways and transcriptional epigenetic regulatory networks crucial for the polarization of macrophages into either the M1 (pro-inflammatory) or M2 (anti-inflammatory) subtypes [43-46].

M1-polarized macrophages, also known as classically activated macrophages, play crucial roles in host defense against viruses and mediate pro-inflammatory responses in acute infections and tumors by producing pro-inflammatory molecules. IFN-γ is an essential inflammatory mediator necessary for the polarization of undifferentiated macrophages into the M1 phenotype. Upon binding of IFN-γ to its receptor, JAK adapters are initially activated, which then leads to the activation of STAT1. Microbial ligands such as lipopolysaccharide (LPS) can activate TLR4, subsequently activating TRIF and MyD88. The pathway regulated by TRIF eventually triggers kinase cascades, leading to the activation of IRF5. Another adapter in TLR4, MyD88, also activates the NF-κB pathway (p50 and p65), a critical transcription factor for M1 macrophage polarization. Furthermore, MyD88 can activate AP-1 through MAPK, which in turn induces M1 characteristics in undifferentiated macrophages [47,48].

In contrast, M2-polarized macrophages, also known as alternatively activated macrophages, are crucial in tissue repair and the treatment of chronic infectious diseases. Similar to M1 macrophages, M2 macrophages primarily produce anti-inflammatory molecules and contribute to immunoregulation. The polarization process of M2 macrophages begins when the cytokines IL-4 and IL-13 bind to the IL-4Rα receptor on the macrophage. This binding triggers the activation of STAT6 through JAK1 and JAK3 signaling, which then translocates into the nucleus to modulate the antiviral immune responses driven by IFN genes. Additionally, other transcription factors, including IRF4 and peroxisome proliferator-activated receptor γ (PPARγ), play roles in mediating immune responses. In this context, STAT6, IRF4, and fatty acid-activated PPARγ regulate a variety of proteins such as arginase 1 (Arg1), Ym1 (or chitinase 3-like 3), resistin-like-α (Fizz1 or Retnla), CCL17, and CD206. Moreover, the interaction of LPS with TLR4 activates the cyclic adenosine monophosphate (cAMP)-responsive element-binding protein (CREB), which in turn activates CCAAT-enhancer-binding protein (C/EBP) and mediates inflammatory responses [47,49].

The balance between M1 and M2 macrophage phenotypes is crucial for effective immune responses and the prevention of pathological conditions. Uncontrolled dysregulation of this balance can lead to various diseases, including chronic inflammatory diseases, autoimmune disorders, and cancer [50]. Despite significant advances, gaps remain in our understanding of the full spectrum of macrophage polarization. One active area of research focuses on the plasticity of macrophages and their ability to transition between M1 and M2 states in response to changing environmental signals [51]. The heterogeneity within the M1 and M2 categories implies that subpopulations of macrophages may exhibit unique functional characteristics based on their microenvironment and stimuli. This plasticity indicates that macrophage phenotypes exist on a continuum rather than as distinct subtypes, which adds complexity to the study of their functions and regulatory mechanisms.

Metabolic reprogramming is a critical factor influencing macrophage phenotype. Recent studies have demonstrated that M1 macrophages primarily utilize glycolysis, while M2 macrophages rely on fatty acid oxidation and oxidative phosphorylation. However, the specific metabolic pathways and their regulatory mechanisms are not yet fully understood [52]. Factors such as hypoxia, nutrient availability, and the presence of other immune cells can significantly alter macrophage behavior [53]. A deeper understanding of these interactions is essential for the development of targeted therapies that can modulate macrophage function in various diseases, thereby enhancing our knowledge of macrophage biology and leading to more effective therapeutic strategies for inflammatory and immune-mediated diseases.

Polarization methods

If standardized methods are developed to initiate the artificial polarization and reprogramming of macrophages, it would be possible to more flexibly regulate inflammatory mediators and the M1/M2 phenotypes, thereby enhancing the immune response to harmful stimuli. However, despite significant advances in macrophage polarization techniques, including those using nanoparticles and chemical agents, achieving a standardized procedure for artificial macrophage polarization in clinical settings remains challenging. These challenges stem from the inconsistency of effects in inducing specific macrophage phenotypes, difficulties in consistent administration, or the complexity of the pathways and cascades involved in the macrophage polarization processes.

Nanoparticles

Nanoparticles have been extensively researched for their ability to artificially alter macrophage phenotypes. Due to their distinctive physicochemical properties, nanoparticles have a wide range of biomedical applications, from therapeutic drugs to disease diagnostics. Once absorbed, nanoparticles are redistributed through the circulatory system to various organs, where they can influence inflammation, allergies, and tumors [54,55]. Studies have shown that various nanoparticles can reprogram and stimulate undifferentiated macrophages to undergo polarization. For instance, Peilin et al. [56] explored the impact of gold nanoparticles (50 nm) on undifferentiated RAW264.7 macrophages and found that the NF-κB and MAPK signaling pathways were inhibited, suggesting induced M2 polarization. Similarly, Lee et al. [57] utilized titanium nanoparticles (30–50 nm) to induce M2 polarization, as indicated by increased Arg1 expression. These findings suggest the potential of nanoparticles for future research aimed at standardizing inflammation treatments. However, the degree of phenotype expression can vary greatly depending on nanoparticle characteristics such as size, crystallinity, aggregation, and surface functionality, which contributes to variability in trial outcomes [58]. Additionally, the ability of nanoparticles to cross physiological barriers may trigger adverse reactions, including size-dependent cytotoxicity, oxidative stress, and organ damage [59]. Therefore, while nanoparticles have potential, the risks of uncontrolled toxicity and inconsistent results must be addressed.

Chemical agents

There are multiple chemical inducers and clarified chemical pathways for stimulating macrophage polarization, suggesting various hypothetical inducers to target for specific polarization. Possibilities range from targeting specific cellular pathways that produce agents directly related to polarization to adding these agents directly into the internal environment. For example, IL-4, an antiinflammatory cytokine, has shown potential in inducing M2 polarization. Celik et al. [60] demonstrated that applying IL-4 to injured mouse nerves shifted F4/80+ macrophages from the M1 phenotype to the M2 phenotype, resulting in the synthesis of opioid peptides such as B-endorphin and Met-enkephalin, which attenuated neuropathy-induced hypersensitivity. Similarly, Yan et al. [61] used leucine as a potential chemical agent to induce polarization among M1 and M2 macrophages. Leucine interacted with the rapamycin complex 1 (mTORC1)/liver X receptor α pathway, thereby reducing the secretion of pro-inflammatory cytokines such as IL-6 and TNF-α, and increasing expression of M2 markers such as Arg1 [62,63]. Despite these promising examples, challenges remain in standardizing and clinically applying chemical agents to manage these complex pathways. One example is selecting and optimizing specific agents for consistent polarization effects is challenging, as are precise dosing and repeated administration, as transient responses are significant limitations.

Overall, while both nanoparticles and chemical agents show promise in inducing macrophage polarization, each approach has significant limitations. Nanoparticles provide versatile biomedical applications but carry risks of cytotoxicity and yield inconsistent results. Chemical agents, such as IL-4, have proven effective in inducing polarization but present challenges in dosing and administration. Addressing these limitations is crucial for developing standardized, effective methods for evaluating macrophage polarization in clinical settings.

PBM-INDUCED MACROPHAGE POLARIZATION

PBM utilizes low-level laser (LLL) therapy that incorporates either coherent or noncoherent light within the red to near-infrared spectrum, ranging from 600 to 1,000 nm. This method has gained considerable interest for its therapeutic potential in clinical settings [10]. The light sources used in PBM are either LLLs or light-emitting diodes (LEDs), chosen for their safety, cost-effectiveness, minimal thermal effects, and the ability to precisely control irradiation parameters. These benefits make PBM an attractive option for addressing the challenges of macrophage polarization, which include inconsistent application, sourcing difficulties, and operational complexity.

Proposed mechanisms of PBM

Despite these promising attributes, the clinical application of PBM remains limited due to an incomplete understanding of its mechanisms at the cellular, molecular, and tissue levels [64]. There are competing theories about the photochemical effects of laser light on cellular metabolism. The most prominent theory suggests that light is absorbed by chromophores within cells [65]. Specifically, CCO in complex IV of the mitochondrial electron transport chain absorbs light wavelengths between 600 and 1,000 nm and releases nitric oxide (NO) [66]. Fig. 3 illustrates how light absorption stimulates the mitochondria, potentially triggering light-sensitive ion channels and initiating downstream cellular cascades [67,68]. This proposed mechanism suggests that PBM may inhibit inflammation by activating SIRT1, thereby exerting anti-inflammatory effects on macrophage polarization and broader inflammatory responses [69].

Another prominent theory suggests that PBM stimulates mitochondria to directly increase ROS levels for a short duration. This activation triggers specific signaling pathways or induces cellular responses [65,70]. These pathways, which include NF-κB, phosphoinositide 3-kinase (PI3K)/Akt, and NF-E2 p45-related factor 2, are associated not only with inflammation control but also with cell survival, apoptosis, and phagocytosis (Fig. 3) [68]. However, due to uncertainties in the field and the absence of conclusive evidence supporting these mechanisms, PBM has not yet been standardized for clinical use.

The role of mitochondrial CCO absorption in PBM suggests that PBM improves mitochondrial function, leading to increased ATP production and modulating ROS levels. These modulations of ROS can influence signaling pathways that govern macrophage polarization, potentially promoting an anti-inflammatory M2 phenotype. Similarly, a transient increase in ROS can activate pathways that initially stimulate M1 polarization before shifting towards M2 as ROS levels normalize. This dual role of PBM in regulating ROS and mitochondrial activity is central to its proposed effects on macrophage behavior.

Effects of PBM on macrophage polarization

Several studies have demonstrated the impact of PBM on macrophage polarization and inflammation control, highlighting its potential to overcome the limitations of other polarization techniques (Table 1) [71-82]. In a recent study by Ma et al. [71], the effects of 808 nm PBM on macrophage polarization were explored in the context of wound healing and the ENT inflammatory response, including conditions such as otitis media. Researchers utilized bone marrow-derived macrophages (BMDMs) polarized to M1 and M2 phenotypes and subjected them to PBM to evaluate changes in macrophage phenotype and function. The findings revealed that PBM treatment significantly increased the expression of M2 markers, such as Arg1 and CD206, in ENT inflammatory conditions, while concurrently decreasing the expression of M1 markers, including inducible nitric oxide synthase (iNOS) and TNF-α. Additionally, PBM-treated macrophages facilitated enhanced wound healing and reduced inflammatory responses in vivo, particularly in treating ENT-specific conditions like chronic sinus infections. This suggests a shift toward an anti-inflammatory, tissue-repairing M2 phenotype. This study underscores the ability of PBM to modulate macrophage polarization, indicating its promising therapeutic potential for managing inflammation and enhancing tissue repair processes.

Moreover, recent research involving stem cells has demonstrated the ability of PBM to induce macrophage polarization. Woo et al. [72] employed 630 nm PBM to prime tonsil-derived mesenchymal stem cells before coculturing them with M1 RAW264.7 macrophages. The study revealed that a 30 J PBM treatment significantly reduced iNOS expression while increasing Arg1 expression, and also led to a notable increase in anti-inflammatory IL-1RA gene expression. These findings suggest that PBM has the potential to enhance macrophage polarization, particularly when used in conjunction with stem cell therapy.

Clinical trials have also demonstrated the effectiveness of PBM in managing macrophage polarization and inflammation. Ryu et al. [73] conducted research on oral ulcers in Sprague-Dawley rats, using infrared pulse lasers (808 nm wavelength, 50 mW power output, 10 mm spot size). The lasers were administered at varying doses (0, 30, 60, and 100 J) every other day over a period of 8 days. Histological assessments and real-time polymerase chain reaction analyses showed that PBM not only enhanced wound healing but also facilitated M2 polarization of macrophages. This suggests that PBM could play a significant role in boosting anti-inflammatory responses in clinical settings.

While the majority of PBM studies have focused on promoting M2 polarization for its anti-inflammatory effects, there is also evidence that PBM can induce M1 polarization. Tian et al. [74] explored the impact of 980 nm PBM on both undifferentiated and M1-polarized RAW264.7 macrophages. They found that PBM not only increased the expression of inflammatory cytokines such as IL-1β, IL-12, Arg1, and TNF-α, but also elevated the mRNA levels of IL-10, IL-12, Arg1, and iNOS. Interestingly, when applied at a concentration of 5 J/cm², PBM enhanced the expression of M1 macrophage markers while simultaneously reducing the levels of IL-6, IL-12, IL-1β, TNF-α, and iNOS. This ability of PBM to steer macrophages towards both M1 and M2 phenotypes underscores its potential to precisely modulate inflammatory responses.

DISCUSSION

The data presented in the table show a consistent trend across multiple studies, indicating that PBM modulates macrophage polarization in ENT-related inflammatory conditions such as chronic rhinosinusitis and otitis media. This modulation involves attenuating M1 phenotypes and enhancing M2 phenotypes. Studies employing wavelengths in the red to near-infrared spectrum, ranging from 630 to 980 nm, have demonstrated a significant downregulation of pro-inflammatory markers including IL-1, TNF-, iNOS, and IL-6 in various macrophage lines such as RAW264.7, THP-1, and primary bone marrow-derived macrophages. For example, PBM at 808 nm in BMDMs significantly reduced the expression of TNF-α and iNOS, which are key drivers of chronic inflammation. Additionally, in vivo wound healing models have shown a reduction in pro-inflammatory markers and accelerated recovery, linking the downregulation of M1 induced by PBM to improved physiological outcomes.

In addition to suppressing M1 markers, PBM consistently upregulates M2-specific markers such as Arg1, CD206, and IL-10 in ENT organ tissues, thereby enhancing healing in conditions like chronic laryngitis and nasal polyps. This shift toward M2 macrophage polarization is consistent across various wavelengths, supporting the hypothesis that PBM facilitates macrophage reprogramming. The dual ability of PBM to suppress pro-inflammatory mediators and enhance anti-inflammatory signaling through pathways such as PI3K/Akt and NF-κB highlights its therapeutic versatility in resolving chronic inflammation and promoting tissue repair.

Moreover, integrating PBM with existing ENT therapies such as antibiotics and surgical interventions could increase treatment efficacy. For instance, combining PBM with antibiotics could target pathogens and modulate the inflammatory response, potentially reducing the need for higher doses of antibiotics and minimizing side effects. Additionally, in surgical settings, PBM may promote faster tissue healing and reduce post-operative inflammation, leading to improved patient outcomes. However, integrating PBM with these therapies requires careful consideration of protocol compatibility and timing to ensure synergistic effects without adverse interactions.

Another critical aspect is the standardization of PBM protocols for clinical use. It is essential to determine optimal dosages, wavelengths, and treatment durations to maximize therapeutic benefits while ensuring safety. Factors including tissue type, depth of light penetration, and individual patient variability must be considered in the development of standardized PBM protocols. Additionally, establishing safety guidelines to prevent potential adverse effects, such as excessive ROS generation or tissue damage, is paramount. Addressing these challenges is necessary to facilitate the widespread clinical adoption of PBM as a reliable treatment modality for ENT-related inflammatory conditions.

Compared with other macrophage polarization methods, such as nanoparticles and chemical agents, PBM offers distinct advantages, including its non-invasive nature and minimal side effects. These features make it particularly suitable for treating ENT conditions like chronic rhinitis and otitis media. In contrast, nanoparticles can pose risks of cytotoxicity, oxidative stress, and organ damage. Meanwhile, chemical agents such as IL-4 require precise dosing and repeated applications, which present challenges in achieving consistent results. PBM’s ability to modulate both M1 and M2 polarization offers flexibility in treating various inflammatory conditions, whereas nanoparticles and chemical agents may often be limited to inducing specific macrophage phenotypes.

However, the studies presented in this review do not fully clarify the extent to which PBM can be utilized to control macrophage polarization in clinical settings, especially in the treatment of chronic ENT diseases. A significant challenge is the variability in the results of these studies, which may stem from the use of different light sources, wavelengths, doses, and treatment protocols, particularly in tissues specific to ENT. For instance, some research has investigated the cellular effects of PBM in vivo on ENT-specific tissues such as the nasal mucosa, while other studies have examined these effects in vitro. This has led to inconsistencies in the expression of specific M1 and M2 markers. Additionally, variations in cell sources and the status of macrophages complicate the support of the conclusions drawn in this review, particularly given the distinct inflammatory microenvironments found in ENT tissues like the middle ear or throat.

Moreover, the precise mechanisms by which PBM influences macrophage cellular processes remain unclear. Competing theories, including the roles of CCO activation and ROS generation, have not been conclusively validated in ENT-related inflammatory conditions. Clarifying how this technology works is essential for its further development in clinical settings, which underscores the need for additional research into noninvasive phototherapy techniques. To augment the potential of PBM as an effective method for macrophage polarization in treating ENT-specific inflammatory diseases, it is critical to address these limitations through rigorous, standardized research that capitalizes on the benefits of PBM while overcoming its challenges.

CONCLUSION

As an integral component of the inherent defense mechanism of the human body, inflammatory responses are crucial in responding to harmful external stimuli and maintaining homeostasis. Numerous studies have investigated the specific mechanisms involved, aiming to decipher the complex pathways and cellular components associated with both pro-inflammatory and anti-inflammatory responses. Macrophages are central to these processes; their function and the substances they secrete vary depending on their phenotype, which changes through a process known as polarization. Precisely controlling this polarization could strengthen defense mechanisms and prevent the additional damage caused by chronic inflammation.

This review has described various methods capable of inducing directed polarization and controlling inflammation, including the use of nanoparticles and chemical compounds. Although these methods successfully achieve directed polarization, they each have limitations, either in controlling their administration or in consistently inducing desired results. In contrast, PBM technology offers the advantage of precise control over irradiation parameters. Additionally, numerous studies have demonstrated the successful M1 and M2 polarization of macrophages using PBM, making it a more practical and promising option for future applications.

Despite the reported success of these studies, research into the relationship between PBM and macrophages remains in its early stages due to the field’s inherent uncertainties. Additionally, the absence of standardized protocols for PBM application complicates the establishment of consistent irradiation parameters. This review emphasizes the need for further research to improve the therapeutic efficacy of PBM in treating ENT-specific inflammation, such as rhinosinusitis and otitis media, as well as exploring other potential clinical applications. More research is essential to accurately assess the capability of this technology to induce M1 or M2 macrophage polarization in ENT organ tissues and to manipulate environmental factors that influence inflammation, thereby aiding in the management of chronic ENT inflammatory conditions.

HIGHLIGHTS

▪ Photobiomodulation (PBM) offers a novel approach to controlling macrophage-driven ENT inflammation.

▪ Inflammatory diseases in ENT organ tissues are modulated by macrophage phenotypes.

▪ PBM shifts macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) states.

▪ Studies show PBM downregulates pro-inflammatory markers and promotes tissue repair.

▪ Future research should standardize PBM protocols for treating chronic ENT diseases.

Notes

Seung Hoon Woo is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

ACKNOWLEDGMENTS

This study was supported by the Dankook Institute of Medicine & Optics. This research was made possible through the support of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (RS-2023-00247651 and NRF-2020R1A6 A1A03043283), the Korea Medical Device Development Fund grant funded by the Ministry of Science and ICT, the Ministry of Trade, Industry, and Energy (MOTIE, Korea), the Ministry of Health & Welfare, and the Ministry of Food and Drug Safety (RS-2020-KD000027), the Regional Innovation Strategy (RIS) through the NRF funded by the MOE (2021RIS-001), the SNUH Lee Kun-hee Child Cancer & Rare Disease Project, Republic of Korea (23C-02300100), and the Technology Innovation Program Development Program (20021987) funded by the MOTIE, Korea.

AUTHOR CONTRIBUTIONS

Conceptualization: KW. Methodology: KW. Validation: YSK, CA, SHW. Formal analysis: KW, YSK. Investigation: KW. Data curation: KW. Visualization: KW. Funding acquisition: SHW. Writing–original draft: KW. Writing–review & editing: YSK, CA, SHW. All authors read and agreed to the published version of the manuscript.

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Fig. 1.

Key intracellular signaling pathways in inflammation. TLRs: toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) on immune cells, initiating an inflammatory response. Activation of TLRs leads to the recruitment of either the myeloid differentiation primary response 88 (MyD88) adaptor protein or a tumor necrosis factor-associated factor (TRAF) family member, which in turn initiates a signaling cascade. This cascade activates transcription factors such as activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB), and interferon regulatory factor 3 (IRF3), which induce the expression of inflammatory cytokines and mediators. NF-κB: The NF-κB pathway involves proteins such as p50, RelA (p65), and inhibitor of NF-κB (IκB). It is activated by various stimuli, including inflammatory cytokines. Activation of the IκB kinase (IKK) complex, which includes IKKα, IKKβ, and IKKγ, leads to the phosphorylation and degradation of IκB proteins. This process releases NF-κB, allowing it to translocate into the nucleus and promote the transcription of pro-inflammatory genes. JAK/STAT: The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway translates extracellular signals into changes in gene expression. Upon ligand binding, which includes cytokines, interferons, and growth factors, receptor-associated JAKs phosphorylate each other, creating docking sites for STAT proteins. The phosphorylated STATs then dimerize and translocate to the nucleus, where they bind to target gene promoters to regulate genes involved in inflammation. MAPKs: mitogen-activated protein kinases (MAPKs), such as extracellular-signal-regulated kinases (ERK1/2), c-Jun N-terminal kinase (JNK), and p38, respond to inflammatory cytokines, osmotic stress, and mitogens. Each MAPK pathway involves a kinase cascade (MAPKKK→MAPKK→MAPK), culminating in the activation of transcription factors that regulate cell survival, differentiation, proliferation, and apoptosis. ERKs are typically activated by mitogens, while JNK and p38 are activated by inflammatory cytokines and stress. TNF, tumor necrosis factor; IL, interleukin; TRIF, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β; IFN, Interferon.

Fig. 2.

Intracellular mechanisms of macrophage polarization. This schematic illustrates various mechanisms of macrophage polarization and the interactive regulation of transcription factors between M1 and M2 signaling. M1 macrophages are induced by interferon (IFN)-γ and lipopolysaccharide (LPS), which activate the nuclear factor-kappa B (NF-κB), Janus kinase (JAK)/signal transducers and activators of transcription (STAT), and mitogen-activated protein kinase (MAPK) pathways. This activation leads to the production of pro-inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-12, nitric oxide (NO), CXC, tumor necrosis factor (TNF), INF, and cyclooxygenase (COX), and triggers subsequent responses. STAT1 is activated by the IFN-γ receptor, while interferon regulatory factor (IRF) 5, NF-κB, and activator protein-1 (AP-1) are activated by toll-like receptor (TLR) 4. Additionally, increased AP1 expression is mediated by cytokine receptors. Conversely, M2 macrophages are promoted by IL-4 and IL-13, which activate the JAK/STAT pathway along with other transcription factors such as STAT6 and IRF4. The fatty acid receptor activates peroxisome proliferator-activated receptor γ (PPARγ), and TLR4 increases cyclic adenosine monophosphate (cAMP)-responsive element-binding protein (CREB) and CCAAT-enhancer-binding protein (C/EBP) levels, leading to the production of anti-inflammatory cytokines, including IL-4, IL-6, IL-10, IL-13, NO, TGF, and arginase 1 (Arg1), and their associated physiological effects. The feedback regulation between M1 and M2 macrophages, controlled by transcription factors and cytokines, plays a crucial role in maintaining the balance of immune response regulation. iNOS, inducible nitric oxide synthase.

Fig. 3.

Proposed mechanisms through which photobiomodulation (PBM) induces macrophage polarization. PBM leads to increased extracellular adenosine triphosphate (ATP) and intracellular Ca²⁺, significantly impacting macrophage polarization. Mechanistically, PBM begins with the absorption of light by mitochondrial cytochrome c oxidase (CCO), which then releases nitric oxide (NO) and boosts ATP production. This increase in ATP secretion enhances its binding to P2X receptors, facilitating Ca²⁺ influx, and to P2Y receptors, triggering the release of Ca²⁺ from endoplasmic reticulum (ER) stores. The resultant rise in intracellular Ca²⁺ activates the protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways, or the phosphoinositide 3-kinase (PI3K)/Akt pathway via calmodulin (CaM), ultimately promoting cell survival and reducing inflammation. Simultaneously, PBM-induced reactive oxygen species (ROS) activate the Src and PI3K/Akt pathways, while the conversion of ATP to cyclic adenosine monophosphate (cAMP) by adenyl cyclase further stimulates protein kinase A (PKA) and Ras, leading to SIRT1 and ERK signaling, which also support anti-inflammatory and cell survival outcomes. PLC, phospholipase C; HSP, heat shock protein; RAS, rat sarcoma.

Table 1.

Summary of the effects of reported PBM on macrophage polarization

Source	Wavelength (nm)	Output (mW)	Beam area (cm²)	Density (J/cm²)	Irradiation (W/cm²)	Cell line	Main results	Reference
LED	630	26.6	3.24	14.76	0.0082	THP-1-derived macrophages from C57BL/6 male mice	• Reduction of lung inflammation	[75]
							• Downregulation of STAT1 phosphorylation
							• No effect on M2 markers
LED	630	10	-	-	-	M1 RAW264.7 cells	• No adverse effects on stemness	[72]
							• Reduced expression of iNOS
							•IncreasedexpressionofArg1andIL-1RA
LED	650	-	0.0225	16,100	0.281	Macrophage-like THP-1 cells	• Reduced expression of IL-6 and IL-8	[76]
Laser	660, 780	15	0.04	7.5	0.28	M1 J774 mouse cells	• Increased expression of IL-6 for 660 nm	[77]
							• Reduced expression of IL-6 for 780 nm
							• Reduced expression of TNF-α, iNOS, and COX-2 with both wavelengths
Laser	660, 808	30	0.028	64	3.58	M0 RAW264.7 cells	• No impact on cell viability	[78]
Laser	660, 808	30	0.028	64	3.58	M0 RAW264.7 cells	• Increased expression of NO for both wavelengths, but higher from 660 nm wavelengths	[78]
Laser	660	30	0.04	1, 4	0.038	M1 and M2 pan macrophages recruited from dorsal wounds in diabetic Wistar rats	• Reduced expression of IL-1β, IL-6, and COX-2	[79]
Laser	660	30	0.04	1, 4	0.038		• Increased expression of CD206 (M2 macrophage receptor) markers	[79]
Laser	808	50	7.065	0.2, 4, 10, 30, 90	0.0071	BV2 microglia cells	• Increased expression of CD86 and iNOS markers	[80]
Laser	808	50	7.065	0.2, 4, 10, 30, 90	0.0071	BV2 microglia cells	• Increased production of NO and expression of CD206 markers	[80]
Laser	808	50	0.01	7.64, 15.28, 25.48	0.63	Oral ulcer macrophages from Sprague-Dawley rats	• Enhanced healing of oral ulcers	[73]
							• Reduced expression of IL-6 and TNF-α
							• Increased expression of IL-10 and TGF-β
Laser	808	50	0.2	150	0.05	M1 and M2 bone marrow macrophage cells	• Reduced neurotoxic polarization of macrophages	[71]
							• Increased expression of Arg1 and CD206 markers
							• Reduced expression of IL-1α, IL-6, and COX-2
Laser	810	150	0.3	-	-	M0 RAW264.7 macrophage cells	• Enhanced neuronal survival and reduced lesion size	[81]
							• Reduced expression of iNOS and TNF-α
							• Increased expression of Arg1 and IL-10
Laser	980	1,000	0.238	0.519	0.04325	M0 macrophages recruited from dorsal wounds in Sprague-Dawley rats	• Reduced expression of CD86 markers, TNF-α and IL-1β	[82]
Laser	980	1,000	0.238	0.519	0.04325		• Increased expression of CD26 markers	[82]
Laser	980	-	2	0.5, 5, 10	-	M0 and M1 RAW264.7 cells	• Reduced expression of IL-6, IL-1β, and TNF-α	[74]
Laser	980	-	2	0.5, 5, 10	-	M0 and M1 RAW264.7 cells	• Increased expression of IL-10, IL-12, Arg1, and iNOS	[74]

PBM, photobiomodulation; LED, light-emitting diode; iNOS, inducible nitric oxide synthase; Arg1, arginase 1; IL, interleukin; TNF-α, tumor necrosis factor α; COX-2, cyclooxygenase-2; NO, nitric oxide; TGF-β, transforming growth factor β.

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