Differential regulatory mechanisms of gut-derived SCFAs (acetate, propionate, butyrate) on the pulmonary TLR2 signaling pathway and their roles in the inflammatory balance of bacterial pneumonia.

Introduction

Bacterial pneumonia, a primary form of lower respiratory tract infection, continues to pose a severe challenge to global public health. Its high morbidity and mortality rates particularly threaten children, the elderly, and immunocompromised individuals. Data from the World Health Organization indicate that pneumonia caused approximately 2.5 million deaths in 2019 alone [1].

An effective host immune response is crucial for successfully combating pneumonia infection. This necessitates not only the rapid recognition and clearance of invading pathogenic microorganisms by the immune system but also the precise regulation of the accompanying inflammatory response to minimize lung tissue damage caused by excessive immune activation and maintain homeostasis. In recent years, the concept of the “Gut–Lung Axis” has emerged, opening new perspectives for understanding the pathogenesis of respiratory health and disease.

This theory emphasizes that the distant gut and its vast, complex internal microbial ecosystem—the gut microbiota—can remotely influence pulmonary physiological functions and immune status through multiple pathways, including immune cell migration, metabolite circulation, and potential neural pathways [2]. Among the numerous gut microbiota metabolites, short-chain fatty acids (SCFAs) have garnered widespread attention due to their diverse biological activities. SCFAs are primarily organic fatty acids produced by the anaerobic fermentation of dietary fiber by commensal gut bacteria, with acetate (C2), propionate (C3), and butyrate (C4) being the most abundant.

They are not only crucial energy sources for intestinal cells but also act as key signaling molecules, and their pleiotropic functions in regulating systemic metabolism, inflammatory responses, and immune responses have been widely confirmed [3].

Growing research evidence indicates that gut-derived SCFAs can be transported to the lungs via blood circulation, directly impacting the local pulmonary immune microenvironment [4]. In the first line of defense against bacterial invasion in the lungs, Toll-like receptor 2 (TLR2) plays an indispensable role in initial recognition and surveillance. TLR2 primarily recognizes key components of Gram-positive bacterial cell walls, including peptidoglycan (PGN) and lipoteichoic acid (LTA), as well as lipopeptides from certain Gram-negative bacteria, mycobacteria, and mycoplasma.

This recognition is crucial for effectively initiating innate immune responses against bacteria [5]. Concurrently, SCFAs themselves possess distinct immunomodulatory capabilities. Research shows that acetate and propionate primarily exert their effects by activating cell surface G protein-coupled receptors GPR41 (FFAR3) and GPR43 (FFAR2), while butyrate is mainly characterized by its ability to effectively inhibit the activity of histone deacetylases (HDACs) within the cell nucleus, although it can also activate certain GPRs [6].

These two primary mechanisms of action (GPR activation and HDAC inhibition) exhibit significant differences in terms of signal initiation site, transmission speed, duration, and downstream effects, providing a potential molecular basis for the differential regulation of the pulmonary TLR2 signaling pathway by acetate, propionate, and butyrate [3].

However, despite our considerable understanding of the existence of the gut–lung axis, the production and basic functions of SCFAs, and the critical role of TLR2 in pulmonary immunity, a core scientific question remains prominent and urgently requires an answer: how exactly do these three major gut-derived SCFAs—acetate, propionate, and butyrate—based on their distinct mechanisms of action, achieve differential and direct regulation of the TLR2 signaling pathway in key pulmonary cells (including alveolar macrophages and epithelial cells)? Furthermore, what specific role does this potential differential regulation play in the complex and dynamic inflammatory balance of bacterial pneumonia—that is, the balance between effective pathogen clearance and limiting tissue damage? Clear experimental evidence directly answering these questions remains extremely scarce in the currently published literature.

Building upon this research background, the primary objective of this review is to synthesize, integrate, and critically analyze current research progress related to the SCFA–pulmonary TLR2 axis. On this basis, we will attempt, based on a profound understanding of molecular mechanisms and limited indirect evidence, to clearly propose and thoroughly discuss a central hypothesis: that acetate/propionate and butyrate may differentially regulate pulmonary TLR2 signaling in terms of temporal dynamics and the nature of their effects, via distinct predominant mechanisms (GPR activation versus HDAC inhibition). Specifically, we speculate that acetate/propionate may predominantly inhibit the excessive activation of TLR2 in the early stages of inflammation through rapid GPR-mediated signaling, whereas butyrate may primarily promote the late-stage resolution of inflammation and restoration of tissue homeostasis through relatively slow but persistent epigenetic reprogramming mediated by HDAC inhibition [4].

This review will follow a logically progressive structure, first delving into the mechanisms of action and regulation of TLR2 and the immunomodulatory pathways of SCFAs. Subsequently, it will elaborate on and substantiate this central hypothesis and its potential underlying molecular mechanisms, and analyze its possible biological significance in the inflammatory balance of pneumonia. Finally, we will summarize the limitations of current research, identify key future research directions and challenges in this field, aiming to deepen the understanding of the complex network of gut–lung axis-mediated pulmonary immune regulation, and to provide the necessary theoretical support for exploring novel microbiome-based strategies for the prevention and treatment of pneumonia.

The gut–lung axis and SCFAs: a long-distance immune dialogue

In recent years, the concept of the “Gut–Lung Axis” has increasingly garnered widespread attention from the scientific community. It reveals the existence of close and bidirectional communication links and mutual influence between two anatomically distant vital organs—the gut and the lungs [7]. The gut is not only the primary site for digestion and absorption, but also harbors the largest and most diverse microbial community in the human body—the gut microbiota.

It is estimated that the total number of these microorganisms is several times that of the human body’s own cells, and their collective genetic repertoire (metagenome) far exceeds the human genome [8]. The gut microbiota are not mere “passengers”; they are deeply involved in various host physiological processes, including the digestion and metabolism of nutrients and the synthesis of essential vitamins [9]. Furthermore, through the metabolites they produce, they profoundly influence the development, maturation, functional maintenance of the host immune system, and overall health status [10].

The gut microbial ecosystem in a healthy human body is composed of trillions of microorganisms, among which bacteria are predominant (mainly belonging to the phyla Firmicutes and Bacteroidetes), but it also includes archaea, fungi (such as Candida species), viruses (especially bacteriophages), and other microbial members [11]. The composition of the gut microbiota varies significantly among individuals and is a dynamic system continuously influenced by a multitude of endogenous and exogenous factors. These factors primarily include: host genetics, mode of birth (vaginal delivery versus Cesarean section), infant feeding patterns (breastfeeding versus formula feeding), age, dietary habits (especially the type and intake of dietary fiber, fats, and proteins), lifestyle (physical activity levels, sleep quality), geographical environmental factors, medication use (especially drugs with significant impact on the microbiota, such as antibiotics and proton pump inhibitors), and whether the host experiences infections, stress, or other disease states [12].

The balanced state of the gut microbiota, known as “eubiosis”, is crucial for maintaining host physiological functions and health.

Production, absorption, and distribution of SCFAs

Short-chain fatty acids (SCFAs) are the main metabolic end-products generated by the gut microbiota (primarily obligate anaerobic bacteria, including Faecalibacterium prausnitzii, Eubacterium spp., Roseburia spp., and certain species within the genus Clostridium) through the anaerobic fermentation of dietary carbohydrates (mainly dietary fibers, such as gums, inulin, resistant starch, fructo-oligosaccharides (FOS), and galacto-oligosaccharides (GOS)) that are indigestible by the host’s own enzyme systems [13]. Among these, acetate (C2), propionate (C3), and butyrate (C4) are the three most abundant SCFAs. Their molar ratio in the colon is typically around 3:1:1, although this ratio can vary depending on individual dietary habits and the specific composition of the gut microbiota [14].

The vast majority of SCFAs produced in the colon are efficiently absorbed by intestinal epithelial cells via specific transporters, including monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter 1 (SMCT1) [15]. Among these, butyrate is the primary energy source preferentially utilized by colonocytes, estimated to meet up to 70% of their energy requirements. In contrast, acetate and propionate are less directly utilized by colonocytes; most of them are absorbed and enter the portal venous system.

Acetate is the SCFA with the highest concentration in peripheral blood circulation, typically reaching micromolar (µM) levels [16]. This is mainly because a relatively small proportion of it is metabolized during its passage through the liver, allowing it to enter systemic circulation and be transported throughout the body, serving as an energy substrate or an important signaling molecule for various peripheral tissues. The concentrations of propionate and butyrate in peripheral blood are relatively lower [17].

This is because they are largely metabolized and cleared during their first pass through the liver (i.e., the hepatic first-pass effect)—propionate is primarily involved in hepatic gluconeogenesis, while butyrate is oxidized to provide energy [18].

Nevertheless, multiple studies have confirmed that SCFAs present in systemic circulation (including acetate, propionate, and butyrate) can indeed reach lung tissue. Researchers have detected the presence of these SCFAs in lung tissue fluid or bronchoalveolar lavage fluid (BALF) [19]. Although the exact physiological concentration range of SCFAs in the lungs (speculated to be in the low micromolar (µM) or even nanomolar (nM) range) and their dynamic changes under different physiological and pathological states remain challenging aspects of research, this undoubtedly provides the necessary material basis for SCFAs to directly act on pulmonary cells and regulate their functions. [13].

It should be emphasized that even relatively low concentrations of SCFAs may exert signaling regulatory effects by activating high-affinity G protein-coupled receptors (GPRs), or they may accumulate in specific local microenvironments (such as inflamed areas) to reach concentrations sufficient to affect HDAC activity. [20].

The concept of the gut–lung axis and its communication pathways

The gut–lung axis describes the complex and intimate network of interactions existing between these two distant organs, the gut and the lungs, under both physiological and pathological conditions. This communication is bidirectional and is primarily mediated through the following key pathways:Immune cell-mediated communication: the gut-associated lymphoid tissue (GALT) is the largest immune organ in the human body. Immune cells activated in the gut by specific antigens or microbial signals (including secretory IgA-producing B lymphocytes, T lymphocytes with specific phenotypes and functions such as regulatory T cells (Tregs) and T helper 17 (Th17) cells, and dendritic cells (DCs)) can migrate via the lymphatic and circulatory systems and selectively home to the bronchus-associated lymphoid tissue (BALT) or lung parenchyma.

These migrated immune cells can directly participate in or finely regulate local immune response processes in the lungs [7].Metabolite-mediated communication: various small molecule substances produced by the gut microbiota during metabolism, such as SCFAs (acetate, propionate, butyrate), secondary bile acids, tryptophan metabolites (including indole and its derivatives), and polyamines, can be absorbed into the bloodstream and transported to the lungs via circulation. These metabolites can directly act on pulmonary immune cells (including macrophages, neutrophils, lymphocytes) or structural cells (including alveolar epithelial cells, airway epithelial cells, fibroblasts), thereby regulating the functions of these target cells by activating specific cell surface receptors (such as GPRs activated by SCFAs) or altering intracellular metabolic states (such as butyrate inhibiting HDACs or affecting cellular energy metabolism) [19].Communication via microbial translocation or component entry into circulation: under certain pathological conditions, when intestinal barrier function is compromised (as seen in inflammatory bowel disease (IBD), severe infections, major surgical trauma, chronic alcoholism, etc.), intestinal bacteria or their structural components (such as lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan (PGN) from Gram-positive bacteria, and bacterial DNA) may breach the intestinal barrier and enter the bloodstream (this process is referred to as ‘bacterial translocation’ and is often associated with ‘dysbiosis’). These circulating microbial-derived molecules can reach the lungs, directly stimulating the pulmonary immune system (e.g., by activating TLRs and other pattern recognition receptors), thereby inducing or significantly exacerbating pulmonary inflammation [21].Neurally mediated communication: the vagus nerve, as an important neural pathway connecting the gut and the brain, as well as the brain and the lungs, may also play a role in gut–lung axis communication.

Signals perceived by the gut (potentially including neurotransmitters produced by the microbiota or metabolites acting on intestinal nerve endings) can be transmitted via the vagus nerve to the central nervous system (Fig. 1). These signals can then, through descending pathways, influence pulmonary inflammatory responses and physiological functions, forming complex neuro-immune regulatory loops [22].Fig.

1The gut–lung axis: production, transport, and distribution of SCFAs

Dietary fibers are fermented by commensal gut microbiota in the intestinal lumen to produce short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate. These metabolites are absorbed by intestinal epithelial cells and enter the portal venous system. The liver exerts a significant “first-pass effect”, where a large proportion of butyrate and propionate is metabolized for energy or gluconeogenesis.

In contrast, acetate largely bypasses hepatic metabolism, becoming the most abundant SCFA in the systemic circulation. Circulating SCFAs eventually traverse the pulmonary vasculature to enter the lung microenvironment, where they act on alveolar macrophages and epithelial cells, thereby establishing a chemical communication bridge between the gut and the lungs.

Association between gut microbiota dysbiosis and lung diseases

A growing body of clinical observations and experimental research evidence strongly indicates that alterations in the composition and function of the gut microbiota (dysbiosis) are closely associated with the occurrence, development, and severity of various lung diseases [19]. Specifically:Asthma: in patients with asthma, reduced gut microbiota diversity is commonly observed, along with alterations in the abundance of specific bacterial groups (some studies have observed a potential decrease in the abundance of Bacteroidetes and an increase in certain members of Proteobacteria). Furthermore, research suggests that the establishment process of the gut microbiota in early life (such as infancy) may have long-term implications for an individual’s future risk of developing asthma [23].Chronic obstructive pulmonary disease (COPD): in patients with chronic obstructive pulmonary disease (COPD), gut microbiota dysbiosis may influence disease progression through multiple pathways, including exacerbating systemic inflammation and affecting the balance of key metabolites like tryptophan, thereby potentially increasing susceptibility to acute exacerbations [3].Cystic fibrosis (CF): in patients with cystic fibrosis (CF), alterations in their gut microbiota have been found to correlate with the types of pathogens causing lung infections and the severity of these infections [24].Pneumonia: in patients with pneumonia (including community-acquired pneumonia (CAP) and ventilator-associated pneumonia (VAP)), the composition and changes in their gut microbiota upon admission or during hospitalization may affect the host’s resistance to lung infection and the post-infection recovery process.

One finding is that drastic changes in the gut microbiota of critically ill patients (often characterized by a sharp decrease in diversity and enrichment of pathogenic bacteria) are considered an important risk factor for the development of VAP [25].Others: furthermore, gut microbiota dysbiosis has also been linked to the pathogenesis of acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), and even lung cancer [26].

Animal model studies have provided further mechanistic support for these clinical associations. Studies have shown that experimentally modulating the gut microbiota (such as by adopting a fiber-rich diet to increase endogenous SCFA production, using broad-spectrum antibiotics to deplete most of the gut microbiota, performing fecal microbiota transplantation (FMT) to reconstruct a healthy microbiota structure, or supplementing with specific prebiotics/probiotics) can significantly alter the intensity of the host’s pulmonary immune response and the final outcome to allergens (such as dust mites), pathogen attacks (such as influenza virus, Streptococcus pneumoniae), or injurious stimuli (such as bleomycin-induced fibrosis) [27]. These studies collectively highlight the crucial role played by the gut microbiota and its metabolites (especially SCFAs), as core mediators of the gut–lung axis, in regulating pulmonary health and disease [28].

Understanding the complex bidirectional communication mechanisms of the gut–lung axis and the pleiotropic roles played by the gut microbiota (especially its key metabolites, SCFAs) therein is crucial for deeply elucidating the pathogenesis of lung diseases. Furthermore, it provides an important theoretical basis and potential targets for the future development of innovative disease prevention and treatment strategies based on targeted modulation of the gut microbiota.

The role of TLR2 in pulmonary innate immunity and bacterial pneumonia

Structure, ligands, and signal transduction of TLR2

Toll-like receptor 2 (TLR2) is one of the key members of the Toll-like receptor (TLR) family and is a type I transmembrane glycoprotein. Its typical structural features include: an extracellular domain rich in leucine-rich repeats (LRRs) responsible for direct ligand recognition; a single transmembrane hydrophobic domain; and an intracellular Toll/Interleukin-1 receptor (TIR) homology domain responsible for recruiting downstream adaptor proteins to initiate signal cascade reactions. Unlike some other TLRs (e.g., TLR4 which recognizes LPS, TLR5 which recognizes flagellin) that can independently recognize ligands, a distinctive feature of TLR2 is that it must form heterodimeric complexes with other TLR family members (primarily TLR1 or TLR6) to effectively recognize and bind its diverse array of ligands [29].

These ligands are mainly pathogen-associated molecular patterns (PAMPs) derived from microorganisms. Specifically: (a) the TLR2/TLR1 heterodimer primarily recognizes triacylated lipopeptides of bacterial origin, which are commonly found in the cell wall or cell membrane structures of various Gram-negative bacteria and Mycobacteria; [30] (b) the TLR2/TLR6 heterodimer has a broader ligand spectrum, primarily recognizing diacylated lipopeptides of bacterial origin. These molecules are characteristic structures of cell membrane lipoproteins from many Gram-positive bacteria (including Staphylococcus aureus) and mycoplasmas (including Mycoplasma pneumoniae).

Additionally, the TLR2/TLR6 heterodimer is involved in recognizing the major component of Gram-positive bacterial cell walls—peptidoglycan (PGN)—and zymosan, a complex polysaccharide derived from fungal cell walls [31]. For important pneumonia pathogens, such as Streptococcus pneumoniae, lipoteichoic acid (LTA) on its cell wall is also a significant agonist for the TLR2/TLR6 heterodimer [32].

When the corresponding PAMP ligand binds to the extracellular LRR domain of the TLR2 heterodimer, it induces a conformational change in the receptor. This promotes the approximation and dimerization of the intracellular TIR domains of the two receptor molecules, thereby forming a signal transduction platform on the inner side of the cell membrane. The core function of this platform is to recruit key downstream adaptor proteins that also contain TIR domains, the most important and initial of which is MyD88 (myeloid differentiation primary response 88) [33].

MyD88 then acts as a crucial adaptor protein to further recruit and activate members of the Interleukin-1 receptor-associated kinase (IRAK) family, such as IRAK4, IRAK1, and IRAK2. A critical step in signal initiation is the autophosphorylation and acquisition of kinase activity by IRAK4 upon recruitment, which then phosphorylates and activates IRAK1 and/or IRAK2. Activated IRAK1/2 dissociates from the receptor-MyD88 complex and forms a complex with another key adaptor protein, TRAF6 (TNF receptor-associated factor 6) [34].

TRAF6 is an important E3 ubiquitin ligase that catalyzes non-degradative K63-linked polyubiquitination of itself and downstream regulatory proteins (such as NEMO/IKKγ). This ubiquitination serves as a crucial signaling regulatory node, enabling the recruitment and activation of the downstream TAK1 (TGF-β-activated kinase 1) kinase complex (which typically includes regulatory subunits such as TAB1/2/3). Activated TAK1 is a key hub in the TLR2 signaling pathway and can phosphorylate and activate at least two major downstream signaling branches [35].

The first is the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) pathway: TAK1 phosphorylates and activates the IKK (IκB kinase) complex (composed of catalytic subunits IKKα, IKKβ, and the regulatory subunit NEMO/IKKγ). The activated IKK complex (mainly IKKβ) then phosphorylates the NF-κB inhibitory protein IκBα. Phosphorylated IκBα is recognized by specific E3 ubiquitin ligases, undergoes K48-linked polyubiquitination, and is subsequently recognized and degraded by the 26S proteasome [36].

The degradation of IκBα exposes the nuclear localization signal (NLS) of the NF-κB transcription factor (most commonly the p50/p65 (RelA) heterodimer), which was previously sequestered in the cytoplasm, allowing its rapid translocation into the nucleus. In the nucleus, NF-κB binds to specific DNA sequences—κB sites—in the promoter or enhancer regions of numerous target genes, recruits transcriptional cofactors, and thereby initiates the transcription of a large number of pro-inflammatory genes. The products encoded by these genes include various cytokines (e.g., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β)), chemokines (e.g., neutrophil chemokines CXCL1/KC, CXCL2/MIP-2; monocyte chemokine CCL2/MCP-1), and adhesion molecules (e.g., ICAM-1, VCAM-1, involved in leukocyte recruitment) [32].These gene products (e.g., TNF-α, IL-6) are core to the pneumonia inflammatory response and, as will be discussed, are key targets for immunomodulation by SCFAs.The second is the MAPK (Mitogen-Activated Protein Kinase) pathway: TAK1 can also phosphorylate and activate MAPK kinase kinases (MAPKKKs, e.g., MEKKs), which in turn, through MAPK kinases (MAPKKs, e.g., MKK3/6, MKK4/7, MEK1/2), sequentially phosphorylate and activate three major terminal MAPK pathway kinases: ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 MAPK.

Once activated, these three MAPK pathways further phosphorylate a large number of downstream substrates within the cell, including various transcription factors. An important target is the AP-1 (Activator protein 1) transcription factor complex, which is typically composed of proteins from the Jun family (e.g., c-Jun) and Fos family (e.g., c-Fos) [37]. Activated MAPKs can phosphorylate Jun and Fos proteins, enhancing their stability and transcriptional activity.

AP-1 then enters the nucleus and binds to AP-1 binding sites on target genes. Typically, AP-1 synergizes with NF-κB to co-regulate the expression of numerous inflammation-related genes as well as other genes involved in cell proliferation, differentiation, and apoptosis [38].

The entire signal transduction process, from ligand binding to the TLR2 heterodimer to the activation of downstream transcription factors NF-κB and AP-1 and the initiation of gene expression, is generally relatively rapid [39]. Key steps (including kinase phosphorylation, IκBα degradation, and transcription factor nuclear translocation) can be completed within a timescale of minutes to an hour. The ultimate activation of the TLR2 signaling pathway leads to a series of important biological effects in immune cells, specifically manifested as: the massive release of inflammatory mediators to recruit and activate other immune cells [40]; enhancement of the cell’s own phagocytic and bactericidal capabilities; and upregulation of major histocompatibility complex (MHC) molecules and co-stimulatory molecules (including CD80, CD86) expression to effectively present antigens to T lymphocytes, thereby initiating and coordinating adaptive immune responses [41].

Furthermore, the TLR2 signaling pathway may also engage in complex crosstalk with other important cellular signaling pathways (such as the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway) and may be involved in the priming (providing signal 1) or activation (providing signal 2) of inflammasomes (such as the NLRP3 inflammasome), further amplifying and modulating the intensity and nature of the inflammatory response [31].

The role of TLR2 in major pulmonary immune cells

TLR2 is expressed on the surface of various immune cells and structural cells in the lungs, and it plays a crucial role in these cells’ execution of their inherent immune defense functions. Specifically, its role is manifested in the following cells:Alveolar macrophages (AMs): as resident phagocytes and primary pathogen-recognizing cells within the alveolar space, AMs express high levels of TLR2. When TLR2 recognizes bacterial PAMPs, AMs are rapidly activated to initiate a series of important defense functions.

These include enhanced phagocytosis (significantly increasing the efficiency of engulfing and internalizing invading bacteria or other particulate matter), production of pro-inflammatory mediators (large-scale synthesis and release of various pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and a range of chemokines, such as CXCL1, CXCL2, CCL2, which collectively recruit circulating neutrophils and other immune cells to the site of infection), production of antimicrobial substances (generating large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through pathways such as NADPH oxidase activation, with these potent oxidants directly killing engulfed bacteria), and upregulation of antigen presentation (enhancing their ability to process and present bacterial antigens to T lymphocytes by upregulating MHC class II molecules and co-stimulatory molecules, thereby effectively initiating adaptive immune responses) [42, 43].Neutrophils: neutrophils are the primary effector cells in combating acute bacterial pneumonia, especially infections caused by pyogenic bacteria. They are rapidly recruited from the bloodstream to infected lung tissue and also express functional TLR2. TLR2 signaling is crucial for neutrophil function, primarily manifested as: promoting chemotaxis and recruitment (TLR2 signaling can enhance neutrophil responsiveness to chemokine gradients, such as CXCL1/2 produced by AMs and other cells, facilitating their migration across the vascular endothelium and into the alveolar space), enhancing bactericidal functions (TLR2 activation significantly boosts key neutrophil bactericidal mechanisms, including phagocytosis, degranulation—the release of stored proteolytic enzymes, antimicrobial peptides like defensins and cathepsin G—and oxidative burst, the rapid production of large amounts of ROS via NADPH oxidase [44], and inducing the formation of neutrophil extracellular traps (NETs) (the TLR2 signaling pathway is also involved in inducing NET release; NETs are web-like structures composed of neutrophil nuclear DNA, histones, and granular proteins that can effectively "trap" and kill bacteria, although their excessive formation can also exacerbate damage to surrounding lung tissue) [45, 46].Dendritic cells (DCs): DCs are crucial bridging cells linking innate and adaptive immune responses.

Both resident and recruited DCs in the lungs express TLR2. Activation of TLR2 signaling can promote DC maturation, endowing them with a stronger ability to initiate adaptive immunity. This includes upregulating co-stimulatory and MHC molecules (mature DCs significantly upregulate the expression levels of cell surface co-stimulatory molecules like CD80, CD86, and MHC class II molecules, which are essential for effective antigen presentation and activation of naive T cells), secreting key cytokines (depending on different stimuli and the microenvironment, mature DCs can secrete specific cytokine profiles, e.g., producing IL-12 to primarily drive Th1 cell differentiation, important for defense against intracellular pathogens, or producing IL-6 and IL-23 to help promote Th17 cell differentiation, important for defense against extracellular bacteria and fungi), and migrating to draining lymph nodes (TLR2 signaling and other maturation signals prompt antigen-laden DCs to migrate from lung tissue to nearby draining lymph nodes, such as hilar lymph nodes, where they present captured antigens to naive T lymphocytes, thereby initiating specific adaptive immune responses) [47].Lung epithelial cells: these include airway epithelial cells lining the airway surface and type I and type II alveolar epithelial cells (AECI, AECII) that constitute the alveolar wall.

They not only form an important physical barrier but also, as sentinel cells of the pulmonary immune system, express TLR2 and can actively participate in immune defense. Their functions include: producing antimicrobial peptides (upon stimulation with TLR2 agonists, lung epithelial cells can synthesize and secrete various antimicrobial peptides, such as β-defensins and LL-37 cathelicidin, which can directly inhibit or kill various bacteria), secreting chemokines (they can produce and release chemokines, such as CXCL8 (IL-8), which is one of the major chemokines for human neutrophils, helping to recruit neutrophils to the site of infection), regulating barrier function (the TLR2 signaling pathway may be involved in regulating the expression and function of tight junction proteins between epithelial cells, thereby affecting the permeability of the alveolar–capillary barrier), and responding to injury (studies have shown that physical stress such as mechanical stretch, for example, excessive stretching of the lungs during mechanical ventilation, can upregulate TLR2 expression levels in lung epithelial cells, suggesting that TLR2 may also be involved in sensing and responding to lung injury, and even influencing subsequent repair and remodeling [48].Other cell types: besides the major immune and structural cells mentioned above, TLR2 expression has also been detected on other cell types in the lungs, and it may be involved in regulating their functions. For example, lung fibroblasts, lung endothelial cells, and certain specific lymphocyte subpopulations (such as γδ T cells) also express TLR2.

TLR2 signaling may influence their inflammatory responses, tissue remodeling behavior, or interactions with other cells [49].

The role of TLR2 in recognizing common pneumonia pathogens

TLR2 is crucial for the host immune system to recognize various common pathogens that cause bacterial pneumonia. Its broad ligand recognition capability enables it to effectively sense the presence of different types of bacteria. For example, TLR2 (usually in conjunction with its heterodimeric partners TLR1 or TLR6) can effectively recognize: (1) Streptococcus pneumoniae: as the most common causative agent of community-acquired pneumonia (CAP), its cell wall contains abundant lipoteichoic acid (LTA) and peptidoglycan (PGN), both of which are potent agonists for the TLR2/TLR6 heterodimer [50].

(2) Staphylococcus aureus: including the increasingly problematic methicillin-resistant Staphylococcus aureus (MRSA), it is an important pathogen in hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). Lipopeptides (which can be di- or tri-acylated) and peptidoglycan (PGN) on its cell wall or cell membrane can be recognized by TLR2/TLR6 or TLR2/TLR1 heterodimers, respectively [31, 32]. (3) Mycoplasma pneumoniae: as a common pathogen causing atypical pneumonia, Mycoplasma pneumoniae lacks a cell wall but its cell membrane is rich in lipoproteins (usually di-acylated), which are agonists for the TLR2/TLR6 heterodimer [51].

(4) Haemophilus influenzae: particularly non-typeable strains (NTHi), it is a common cause of acute exacerbations in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia. Although its main immunostimulant is lipopolysaccharide (LPS, primarily recognized by TLR4), some of its lipooligosaccharide (LOS) variants or outer membrane proteins (OMPs) may also act through the TLR2 signaling pathway or modulate TLR4 signaling [52]. (5) Mycobacteria species: for example, Mycobacterium tuberculosis, which causes tuberculosis, has an extremely complex cell wall structure rich in various unique lipids and glycolipids, such as lipoarabinomannan (LAM) and various other lipopeptides.

These components are primarily recognized by host cells via the TLR2/TLR1 heterodimer [53].

In addition to its core role in recognizing bacterial PAMPs, recent studies have also reported that TLR2 may be involved in recognizing components of certain viruses or virus-induced molecular patterns. For example, the fusion (F) protein of respiratory syncytial virus (RSV) or the spike (S) protein of certain coronaviruses (such as SARS-CoV-2) may interact with TLR2 and activate downstream inflammatory signaling pathways, but the specific mechanisms and physiological significance in this regard are still under investigation. Furthermore, TLR2 (often in synergy with other pattern recognition receptors like TLR6 or Dectin-1) is also involved in recognizing cell wall components of certain fungi, such as zymosan, which is widely found in the cell walls of yeasts and some fungi [54, 55].

These findings suggest that the role of TLR2 in pulmonary anti-infective immunity may be broader than initially thought, encompassing the recognition of bacteria, some viruses, and fungi. However, it must be pointed out that compared to its core and well-defined role in recognizing bacterial PAMPs (especially lipopeptides, PGN, LTA), the specific mechanisms, signaling pathway details, and functional contributions of TLR2 in recognizing viral and fungal PAMPs and mediating corresponding immune responses in pneumonia still require more in-depth research to be fully elucidated [56, 57].

The importance of TLR2 in the inflammatory balance of bacterial pneumonia

The TLR2-mediated early immune response is crucial for the timely initiation of defense and for limiting bacterial replication and dissemination. Multiple studies have confirmed that upon infection with TLR2-dependent pathogens (such as Streptococcus pneumoniae or Staphylococcus aureus), TLR2 gene knockout mice often exhibit decreased bacterial clearance, increased pulmonary bacterial load, and increased mortality [32], highlighting the necessity of TLR2 in effective antibacterial defense. However, the key to maintaining pulmonary immune homeostasis lies in balance.

TLR2 signaling not only initiates defense, but its intensity and duration also profoundly influence the outcome of inflammation. On one hand, TLR2 signaling can, in some cases, help shape protective adaptive immune responses. For example, in anti-Toxoplasma gondii infection, TLR2 signaling has been shown to induce essential Th1-type immune responses while suppressing detrimental Th2-type immune responses [58].

On the other hand, to prevent sustained or excessive activation of the TLR2 signaling pathway from causing catastrophic tissue damage, the body has evolved multi-layered, precise negative regulatory mechanisms. These mechanisms act at different levels of the signaling pathway, exerting fine-tuned inhibition and modulation of the signal [59]. These regulations are primarily achieved through the following aspects:Receptor-level regulation: this includes (a) interference and promotion of degradation by endogenous molecules: for example, pulmonary surfactant proteins SP-A and SP-D (belonging to the collectin family) not only participate in maintaining alveolar surface tension but also possess important immunomodulatory functions.

They can directly bind to the extracellular domain of TLR2, thereby physically interfering with the binding of TLR2 to bacterial ligands (such as LTA), or promote the endocytosis and subsequent lysosomal degradation of the TLR2 receptor complex, thus effectively inhibiting the initiation of downstream signaling and inflammatory responses (such as TNF-α production) [60]. (b) Soluble decoy receptors: cells can produce a soluble form of TLR2 (sTLR2), which lacks the transmembrane domain and the intracellular TIR domain. sTLR2 released into the extracellular fluid can act as a "decoy" receptor to competitively bind bacterial ligands, thereby preventing ligands from binding to functional TLR2 on the cell membrane, exerting a negative regulatory effect [61].Regulation at the intracellular signaling level: this is mainly achieved through inhibitory adaptor proteins and kinases. There are various key negative regulatory proteins within the TLR signaling pathway.

They are induced or activated after signal activation, forming negative feedback loops to limit the intensity and duration of the signal. These proteins include (a) TOLLIP (toll interacting protein): in the resting state, TOLLIP is typically associated with IRAK kinases, playing a role in preventing their premature activation and thereby dampening excessive TLR signaling [62]. (b) IRAK-M (IL-1 receptor-associated kinase M): is primarily expressed in monocytes/macrophages, and its expression is induced by TLR signaling.

IRAK-M is a member of the IRAK family that lacks kinase activity. It can stably bind to the MyD88-IRAK complex but cannot effectively phosphorylate downstream substrates. Instead, it prevents the association and dissociation of active IRAK1/4 with TRAF6, thereby specifically inhibiting MyD88-dependent downstream signaling (NF-κB and MAPK activation) in the TLR pathway, and is one of the key molecules in inducing the phenomenon of “endotoxin tolerance” [63].

(c) SOCS (suppressor of cytokine signaling) family proteins: such as SOCS1 and SOCS3, their expression is also induced by TLR signaling (as well as other cytokine signals). SOCS proteins can exert inhibitory effects through multiple mechanisms, such as by directly binding to and inhibiting key kinases in signaling pathways (like JAKs, which primarily act in cytokine pathways but may also affect TLR signaling crosstalk), or by acting as components of E3 ubiquitin ligase complexes to mediate the ubiquitination and proteasomal degradation of signaling molecules (such as IRAK1, TRAF6, and MyD88 itself), thereby terminating the signal [61]. (d) A20 (also known as TNFAIP3): A20 is a key ubiquitin-editing enzyme with dual deubiquitinase and E3 ubiquitin ligase activities, and its expression is strongly induced by TLR signaling.

A20 can terminate TRAF6-dependent signaling by removing K63-linked polyubiquitin chains (required for signal activation) from TRAF6 and conjugating K48-linked polyubiquitin chains (mediating proteasomal degradation). It can also inhibit the NF-κB signaling pathway through similar or other mechanisms (such as directly inhibiting the IKK complex). Loss of A20 function leads to severe autoinflammatory diseases, highlighting its central role in controlling inflammation [64].

(e) Other inhibitors: these include SIGIRR (single immunoglobulin IL-1-related receptor), a transmembrane protein whose intracellular domain can interfere with the formation of TLR signaling complexes [65]; MyD88s (MyD88 short), an alternative splice variant of MyD88 that lacks the intermediate domain, cannot effectively transduce signals, and can act as a dominant-negative regulator [66]; and some phosphatases, such as MKP-1 (MAP kinase phosphatase-1), which can specifically dephosphorylate and inactivate MAPKs (p38, JNK), thereby inhibiting MAPK pathway-mediated inflammatory responses [67].Pharmacological intervention: clinically widely used glucocorticoids are potent immunosuppressants, and part of their anti-inflammatory effect is also achieved by inhibiting the TLR signaling pathway. Glucocorticoids can broadly inhibit multiple stages of the TLR signaling pathway through genomic mechanisms (binding to glucocorticoid receptors, inhibiting pro-inflammatory gene transcription, inducing the expression of anti-inflammatory genes like MKP-1) and non-genomic mechanisms (rapidly interfering with signaling pathway proteins, such as inhibiting NF-κB activity), which may include downregulating the expression level of TLR2 itself [68].

Dysregulation of TLR2 signaling, whether insufficient signaling (which can lead to increased susceptibility to specific pathogens, e.g., TLR2-deficient mice are more sensitive to Toxoplasma gondii infection [58], or TLR2 polymorphisms in some human populations are associated with infection susceptibility) or excessive signaling, will lead to pathological states. In particular, sustained or excessive activation of the TLR2 signaling pathway is a key driver of severe pulmonary inflammatory damage (such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)) [31]. Excessive release of pro-inflammatory mediators (i.e., a “cytokine storm”) leads to uncontrolled inflammation, causing massive neutrophil infiltration, destruction of the alveolar–capillary barrier, and pulmonary edema, thereby severely threatening life.

In systemic inflammatory states such as sepsis, the continuous activation of TLR2 by bacterial components in the circulatory system also plays an important role in the development and progression of multiple organ dysfunction (including ARDS). The relationship between TLR2 activation and apoptosis is also quite complex. In some cases, it can induce apoptosis [69], which may both help clear infected cells and exacerbate tissue damage due to excessive apoptosis (especially of epithelial and endothelial cells).

Therefore, the TLR2 signaling pathway must be precisely and dynamically regulated. Its regulation is a precise dynamic feedback process: it needs to be appropriately enhanced in the early stages of infection to initiate defense mechanisms, and then promptly attenuated with the activation of negative feedback mechanisms to limit inflammatory intensity, promote inflammation resolution, and facilitate tissue repair. A thorough understanding of how endogenous factors (such as genetic background, age) and exogenous factors (such as gut microbiota and their metabolites, SCFAs) affect this delicate balance of TLR2 signaling is of paramount importance for comprehensively elucidating the pathophysiology of pneumonia and for exploring novel targeted therapeutic strategies aimed at "modulating" rather than completely "blocking" TLR2 signaling.

Immunomodulatory mechanisms of SCFAs: GPRs and HDAC inhibition

SCFAs, as key metabolites of the gut microbiota, are extensively involved in host immunomodulation through multiple mechanisms. Among these, the activation of G protein-coupled receptors (GPRs) and the inhibition of histone deacetylases (HDACs) are two of the most extensively studied and interconnected major pathways. Understanding the characteristic differences between these two mechanisms is crucial for elucidating how SCFAs differentially affect TLR2 signaling.

The G protein-coupled receptor (GPR) pathway: rapid cell surface signaling

SCFAs can act as ligands for specific GPRs. These receptors are primarily expressed on the surface of immune cells and intestinal epithelial cells, mediating rapid transmembrane signal transduction. The main SCFA receptors include:

(1) GPR43 (also known as FFAR2): it has a high affinity for acetate and propionate, and a relatively lower affinity for butyrate. It is expressed in various immune cells (such as neutrophils, monocytes/macrophages, eosinophils, some T cell subsets), as well as in adipocytes and intestinal epithelial cells. (2) GPR41 (also known as FFAR3): it has a high affinity for propionate and butyrate, and a lower affinity for acetate.

It is mainly expressed in intestinal endocrine cells, adipocytes, and sympathetic nerve endings, and is also found in some immune cell subsets. (3) GPR109A (also known as HCAR2): this receptor primarily recognizes butyrate and niacin (vitamin B3) [70] and is expressed in intestinal epithelial cells, adipocytes, and various immune cells such as macrophages and dendritic cells. (4) Olfr78 (mouse ortholog)/OR51E2 (human ortholog): this is a receptor for acetate, initially discovered in the olfactory system.

Subsequent research has shown its expression in vascular smooth muscle cells, renal juxtaglomerular cells, and other locations, but its specific role in immune cells remains unclear [71].

In terms of signal transduction characteristics, signals triggered by GPR activation typically feature rapidity and diversity. Since GPRs are located on the cell membrane surface, ligand binding can rapidly (usually within seconds to minutes) activate downstream signaling pathways, leading to immediate cellular responses. These GPRs can couple to different types of heterotrimeric G proteins (mainly Gi/o and Gq/11), thereby activating or inhibiting various downstream effectors and signaling pathways, producing diverse biological effects.

GPR43 and GPR41 mainly couple to Gi/o proteins, and by inhibiting the activity of adenylyl cyclase (AC), they lead to a decrease in the intracellular levels of the second messenger cyclic AMP (cAMP) [72]. cAMP typically promotes inflammatory responses through pathways such as activating protein kinase A (PKA) (e.g., by inhibiting the production of certain anti-inflammatory factors or promoting the function of certain pro-inflammatory cells). Therefore, the reduction in cAMP levels is considered one of the important mechanisms for GPR-mediated anti-inflammatory effects. Concurrently, GPR43 and GPR41 can also activate the MAPK pathway (ERK, p38, JNK) and the PI3K/Akt pathway through G protein-dependent (e.g., coupling to Gq/11) or -independent (e.g., via β-arrestin recruitment) pathways [73].

These pathways play complex roles in cell proliferation, survival, metabolic regulation, and certain inflammatory responses (such as cytokine production), and their ultimate effects exhibit high cell-type and stimulus context dependency. Furthermore, Gq/11-coupled GPRs (such as GPR43 expressed in specific cell types or conditions) can activate phospholipase C (PLC), which in turn hydrolyzes PIP2 to produce IP3 and DAG. IP3 acts on IP3 receptors on the endoplasmic reticulum, causing an increase in intracellular Ca2 + concentration; while DAG activates protein kinase C (PKC).

Elevated calcium signaling and activated PKC signaling will further fine-tune various cellular functions [73].

Important interactions or crosstalk may exist between GPR signaling pathways and TLR signaling pathways. The decrease in cAMP levels mediated by GPR43/GPR41 activation may indirectly inhibit the expression of certain TLR-induced pro-inflammatory genes by affecting PKA activity or other cAMP-dependent signaling pathways. Moreover, the activation of pathways like MAPK/Akt by GPRs may also share certain downstream signaling nodes with TLR pathways, thereby modulating the intensity, duration, or final biological effects of TLR signaling.

Notably, a recent study in human lung cancer cell lines clearly revealed a direct antagonistic effect between FFAR2 (i.e., GPR43, for which acetate and propionate are major agonists) and the TLR2 signaling pathway: activation of FFAR2 can significantly inhibit NF-κB activation induced by TLR2 (and TLR3) agonists by suppressing the cAMP-dependent AMPK–TAK1 signaling axis [5]. This finding provides significant molecular mechanistic support for the possibility that GPRs, particularly GPR43, may negatively regulate TLR2-driven pro-inflammatory responses by directly intervening with TAK1, a key kinase upstream of NF-κB in the TLR signaling pathway.

Based on the differing affinities of various SCFAs for their receptors (acetate and propionate are major agonists for GPR43, while propionate and butyrate are major agonists for GPR41), it can be hypothesized that: through the rapid GPR-mediated signal regulatory mechanism, acetate and propionate may play a more prominent role in the rapid suppression of early inflammatory responses. Their mechanisms include rapidly limiting excessive neutrophil chemotaxis or the burst release of early pro-inflammatory cytokines, primarily achieved through GPR43-mediated inhibition of key pro-inflammatory signaling pathways such as TLR2–TAK1–NF-κB.

The HDAC inhibition pathway: relatively slow but persistent epigenetic regulation

In addition to signaling through cell surface receptors, short-chain fatty acids (SCFAs), particularly butyrate, can also enter the cell interior via specific transporters (e.g., monocarboxylate transporters MCT1/SMCT1) or free diffusion, and be further transported into the nucleus, where they directly act on key nuclear enzymes—histone deacetylases (HDACs) [74]. HDACs belong to an important class of epigenetic modifying enzymes. Their primary function is to catalyze the removal of acetyl groups from the N-terminal lysine residues of histones (mainly core histones H2A, H2B, H3, and H4).

Histone deacetylation typically leads to a more compact chromatin structure, forming a heterochromatin state [75]. This state makes it difficult for transcription factors and the entire transcriptional machinery to effectively bind to DNA, thereby generally suppressing gene transcriptional activity.

The signal transduction characteristics of the HDAC inhibition pathway are mainly reflected in the following aspects: firstly, regarding the inhibition of HDAC enzyme activity, butyrate is widely recognized as an effective, non-selective inhibitor of class I (HDAC1, 2, 3, 8) and class IIa (HDAC4, 5, 7, 9) HDACs [76]. This means that butyrate can simultaneously inhibit the activity of multiple HDAC subtypes. Although acetate and propionate also exhibit some HDAC inhibitory capacity, they typically require higher concentrations to achieve an inhibitory effect comparable to that of butyrate.

For example, many in vitro studies use millimolar (mM) levels of butyrate to observe significant HDAC inhibition, a concentration much higher than physiological levels in peripheral blood (µM range) but potentially relevant to concentrations in the intestinal lumen or local inflammatory microenvironments. Therefore, under physiological concentrations, butyrate is generally considered the primary endogenous HDAC inhibitor. Secondly, the core of this pathway lies in its epigenetic regulatory capacity [76].

Inhibition of HDAC activity disrupts the dynamic balance between histone acetylation and deacetylation within the cell, leading to a significant increase in the overall acetylation levels of histones, particularly at specific lysine sites on histones H3 and H4 (e.g., H3K9ac, H3K27ac, H4K8ac, H4K16ac), resulting in histone hyperacetylation. Hyperacetylated histones promote a more relaxed and open chromatin structure, forming a euchromatin state. This greatly enhances the accessibility of transcription factors (such as NF-κB, AP-1, STATs, etc.) to promoter and enhancer regions on the DNA, thereby promoting or inhibiting (depending on the specific gene and regulatory element characteristics) the transcription initiation and elongation processes of specific genes.

Notably, the substrates of HDACs are not limited to histones [77]; the acetylation status of many non-histone proteins (including important transcription factors, signaling proteins, cytoskeletal proteins, etc.) is also regulated by HDACs. Therefore, the inhibitory effect of butyrate on HDACs may also directly alter the acetylation levels of these non-histone proteins, thereby affecting their stability, subcellular localization, or interactions with other molecules, thus fine-tuning their biological functions. Furthermore, gene expression changes mediated by HDAC inhibition are typically a relatively slow process.

As this process involves multiple precise steps, including complex chromatin remodeling, precise recruitment of transcriptional machinery, effective mRNA synthesis and processing, and subsequent protein translation and modification, its final functional effects often require several hours or even longer to fully manifest. However, compared to rapid signals mediated by cell surface receptors, gene expression changes driven by epigenetic alterations may have more persistent characteristics. Even after the inducing signal (such as butyrate) disappears, the effects produced may be maintained for a considerable period, reflecting certain features of epigenetic memory [78].

Additionally, HDACs are involved in regulating the expression of hundreds to thousands of genes within the cell. These genes are widely involved in various crucial biological processes, including inflammatory responses, immune responses, cell cycle regulation, cell differentiation, apoptosis, and metabolic homeostasis. Therefore, butyrate, as a broad-spectrum pan-HDAC inhibitor, often has global effects, capable of extensively remodeling the cell’s overall gene expression profile [79].

The specific mechanisms by which butyrate regulates immune responses and inflammation through HDAC inhibition have been preliminarily elucidated in various lung-related cells and disease models. For example, in a mouse model of acute lung injury induced by LPS (a TLR4 agonist, whose downstream signaling shares commonalities with TLR2), pretreatment with butyrate significantly reduced lung tissue pathological damage, decreased neutrophil infiltration, and inhibited the production of key pro-inflammatory cytokines (such as TNF-α and IL-1β), and markedly suppressed the nuclear translocation and activation of the NF-κB p65 subunit [80]. In allergic asthma models, intranasal administration of butyrate can inhibit NF-κB p65 activity by inhibiting HDAC1 (affecting H3K9 acetylation), thereby ameliorating airway inflammation and pulmonary fibrosis [81].

Furthermore, butyrate can also directly affect the function of specific immune cells via the HDAC inhibition pathway: it can effectively reduce the migration, adhesion, and survival of human eosinophils, an effect that is independent of GPR41/43 but is associated with increased histone acetylation levels [82]. Meanwhile, butyrate (and propionate) has also been reported to influence macrophage polarization towards the M2 phenotype (generally considered anti-inflammatory or pro-repair) through HDAC inhibition and/or GPR43 activation, although its specific effects (promoting or inhibiting M2) may vary in different disease contexts [83]. These studies collectively indicate that butyrate-mediated HDAC inhibition can extensively intervene in key aspects of pulmonary inflammatory processes, including the activity of key transcription factors (like NF-κB), the balance of pro-/anti-inflammatory cytokines, and the functional state of immune cells (such as macrophages and eosinophils).

Finally, HDAC inhibition can modulate the downstream effects of TLR signaling pathways through various complex mechanisms, including: regulating the expression levels of TLR signaling pathway components or their regulatory factors (e.g., HDAC inhibition may alter the gene expression of TLR receptors themselves, key adaptor proteins, kinases, or their important negative regulators such as IRAK-M, A20, SOCS, etc.); regulating the expression of downstream inflammation-related genes (this is one of the primary modes), whereby HDAC inhibition can fine-tune their transcriptional activity under TLR2 signaling stimulation by altering the chromatin accessibility of promoter or enhancer regions of key inflammatory genes, such as pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-12), anti-inflammatory cytokines (IL-10, TGF-β), and various chemokines and adhesion molecules [63]. For instance, numerous studies have shown that butyrate, through HDAC inhibition, can significantly enhance the expression of the key anti-inflammatory gene IL-10, while under certain specific conditions, it can suppress the expression of some key pro-inflammatory genes like TNF-α. However, it must be emphasized that this regulation is not a simple "all-or-none" pattern but is highly dependent on the specific cell type, the type and intensity of the stimulus, the timing of action, and the complex integration state with other signaling pathways) [75]; and regulating the direct activity of transcription factors (butyrate’s inhibition of HDACs may also directly affect the acetylation status of key transcription factors, such as the p65 subunit of NF-κB, thereby altering their DNA binding ability, transcriptional activation potential, or interaction with transcriptional co-activators/co-repressors, and consequently influencing their precise regulation of TLR downstream target gene expression) [84].

Based on the biochemical properties of different SCFAs, butyrate is recognized as the most potent endogenous HDAC inhibitor. Although acetate and propionate also possess some HDAC inhibitory activity, within physiological concentration ranges, the epigenetic regulatory role played by butyrate via the HDAC inhibition pathway is generally considered more important and prominent. This characteristic strongly suggests that butyrate may play a more critical and lasting regulatory role in the mid–to–late stages of the inflammatory response by reprogramming cellular gene expression, for example, by promoting the smooth resolution of inflammation and the subsequent effective repair and functional remodeling of tissues (Fig.

2).Fig. 2Molecular mechanisms: differential regulation of TLR2 signaling by SCFAs

Activation of the Toll-like receptor 2 (TLR2) heterodimer by bacterial ligands (e.g., LTA, lipoproteins) triggers the MyD88-dependent signaling cascade, involving IRAKs, TRAF6, and TAK1. This leads to the activation of MAPKs and the nuclear translocation of NF-κB, resulting in the transcription of pro-inflammatory cytokines (e.g., TNF-α, IL-6).SCFAs modulate this pathway via two distinct mechanisms: A GPR-mediated rapid signaling: acetate and propionate bind to cell surface G protein-coupled receptors (GPR43/FFAR2, GPR41/FFAR3). This interaction rapidly interferes with TLR2 downstream signaling, potentially by inhibiting the TAK1–NF-κB axis or modulating intracellular cAMP levels, acting as an early "brake" on inflammation.

B HDAC inhibition-mediated epigenetic remodeling: butyrate enters the cell (e.g., via MCT1) and acts in the nucleus as a histone deacetylase (HDAC) inhibitor. By preventing histone deacetylation, butyrate promotes chromatin relaxation (hyperacetylation), which suppresses NF-κB p65 activity and induces the expression of anti-inflammatory genes (e.g., IL-10), facilitating inflammation resolution and tissue repair.

The role of the SCFA–TLR2 axis in the inflammatory balance of bacterial pneumonia: literature evidence and analysis

The pathophysiological process of bacterial pneumonia is dynamic and complex, requiring the host immune system to strike a delicate balance between effectively clearing invading pathogens and limiting lung tissue damage caused by excessive inflammation. TLR2, as a key receptor for recognizing various pneumonia pathogens (as detailed in Sect. “The role of TLR2 in recognizing common pneumonia pathogens”), its signal activation is crucial for initiating an early and effective innate immune defense [85].

However, if uncontrolled, it can lead to catastrophic inflammatory consequences. Gut-derived SCFAs, as important immunomodulators, influence immune cell function through mechanisms such as GPR activation and HDAC inhibition (as detailed in Sect. “Immunomodulatory mechanisms of SCFAs: GPRs and HDAC inhibition”), and may differentially regulate the TLR2 signaling pathway (as previously hypothesized).

This chapter aims to integrate existing knowledge and the previously proposed hypotheses to explore how the SCFA–TLR2 axis, as a whole, might play a key role in the inflammatory balance of bacterial pneumonia [86]. It is emphasized again that because literature directly investigating the association between SCFAs, TLR2 signaling, and the delicate inflammatory balance in pneumonia (including pathogen clearance efficiency versus tissue damage extent, or inflammation resolution kinetics) is extremely limited, the following analysis is primarily based on the integration of existing evidence and mechanistic speculation [87].

The importance of maintaining inflammatory balance

In bacterial pneumonia, an ideal host response should possess the following characteristics: (1) early, rapid, and effective pathogen clearance, which requires the timely recruitment and activation of phagocytic cells such as neutrophils and macrophages, and the production of moderate levels of pro-inflammatory cytokines (including TNF-α, IL-1β) to enhance bactericidal capacity and further recruit immune cells [88]; (2) effective control and resolution of late-stage inflammation. After the pathogen is effectively controlled, the inflammatory response needs to be promptly suppressed and resolution programs initiated to avoid irreversible damage to alveolar structure and gas exchange function caused by persistent release of inflammatory factors and immune cell infiltration [89]. This involves the production of anti-inflammatory cytokines (including IL-10), suppression of pro-inflammatory signals, and activation of tissue repair mechanisms.

TLR2 signaling is indispensable for initiating the early response, but its intensity and duration must be precisely regulated to serve the overall goal of inflammatory balance.

“Possible” roles of the SCFA–TLR2 axis in inflammatory balance

Based on the known role of TLR2 in pneumonia and the potential differential regulatory mechanisms of SCFAs on TLR2 signaling (the central hypothesis discussed in this review), we can speculate that the SCFA–TLR2 axis may influence key aspects of the inflammatory balance in pneumonia through the following ways:Regulating neutrophil recruitment and activation: TLR2 signaling is one of the important signals driving neutrophil chemotaxis and activation (including oxidative burst). If acetate/propionate can inhibit key upstream nodes of the TLR2 signaling pathway (e.g., TAK1 activation, thereby inhibiting NF-κB signaling [5] through GPR-mediated mechanisms (especially GPR43/FFAR2), this may help limit initial excessive neutrophil infiltration and activation, preventing early excessive tissue damage. Meanwhile, butyrate, through its HDAC inhibitory activity, has been shown to inhibit LPS (a TLR4 agonist, but its downstream pathway shares the NF-κB pathway with TLR2)-induced neutrophil infiltration and NF-κB activation [80], and to regulate NF-κB p65 via HDAC1 inhibition in asthma models [81].

These effects suggest that butyrate may influence neutrophil dynamics and inflammation resolution in the later stages of inflammation, although its direct effects under TLR2-specific stimulation still require more research.Influencing macrophage polarization and function: TLR2 activation can induce macrophages to produce large amounts of pro-inflammatory factors, promoting M1 polarization. Butyrate, through HDAC inhibition, is known to promote the production of the anti-inflammatory factor IL-10 and may influence macrophage polarization towards the M2 type (anti-inflammatory/repair type) [83]. One study showed that butyrate and propionate can regulate M2 macrophage polarization through GPR43 activation and/or HDAC inhibition, although in an OVA-induced asthma model, butyrate and propionate inhibited M2 polarization, suggesting that their effects are disease- and microenvironment-specific [83].

Furthermore, a recent study showed that butyrate can ameliorate LPS-induced acute lung injury by inhibiting the TLR2/MyD88/NF-κB pathway and oxidative stress [90]. Although this was in an LPS (TLR4) model, it clearly linked the protective effect of butyrate to the inhibition of the TLR2 signaling pathway. This provides an important clue that butyrate may directly regulate gene expression downstream of TLR2 signaling in pulmonary macrophages (Hypothesis 2), changing their functional phenotype from predominantly pro-inflammatory in the early stage to anti-inflammatory and tissue repair-oriented in the later stage.Regulating the pro-/anti-inflammatory cytokine balance: TLR2 signaling primarily drives the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β).

The early inhibitory effect of acetate/propionate on TLR2–TAK1–NF-κB signaling mediated by GPR43 [5] may directly reduce the peak levels of these pro-inflammatory cytokines. The late-stage remodeling effect of butyrate, particularly its ability to promote IL-10 expression through HDAC inhibition and to inhibit LPS-induced TNF-α and IL-1β production [80], may play a key anti-inflammatory role in the later stages of inflammation, helping to restore cytokine balance.Influencing the Th1/Th2/Treg balance: TLR2 signaling, in some cases, helps induce Th1-type immune responses (e.g., in anti-Toxoplasma gondii infection [58]). SCFAs (especially butyrate) are known to significantly promote Treg cell differentiation and function through HDAC inhibition [91], and may influence the Th1/Th2 balance.

Therefore, it is speculated that SCFAs may, by directly regulating T cell differentiation and indirectly modulating TLR2 signaling and its downstream cytokine environment (including IL-12, IL-10), collectively influence the balance of T cell subsets in adaptive immunity. This is crucial for controlling inflammatory intensity and promoting long-term immune homeostasis.Regulating lung epithelial barrier function: TLR2 is expressed in lung epithelial cells and is involved in maintaining barrier function and responding to pathogens. Stresses such as mechanical stretch can upregulate TLR2 expression in epithelial cells [48].

Whether and how SCFAs affect airway barrier integrity, mucus secretion, or the production of antimicrobial peptides (e.g., LL-37, whose expression is regulated by vitamin D3 and TLR2 signaling and can be synergistically enhanced by HDACi like butyrate [92]) by modulating epithelial cell TLR2 signaling are key aspects for maintaining pulmonary defense and preventing pathogen dissemination that warrant in-depth investigation.

Analysis of hypothesized differential roles

Our central hypothesis posits two main components regarding the differential roles of SCFAs: first (Hypothesis 1), that acetate/propionate mediate early "buffering" via GPRs, and second (Hypothesis 2), that butyrate mediates later "remodeling" and "pro-resolution" effects via HDAC inhibition. Combining these (Hypothesis 1 and Hypothesis 2), we can further speculate on the different emphasized roles that various SCFAs might play in maintaining the inflammatory balance in pneumonia, primarily including:Acetate/propionate (GPR-mediated) may predominantly play an early “buffering” role. Through rapid inhibition of the TLR2 signaling pathway (specifically by inhibiting TAK1 activation, thereby inhibiting downstream NF-κB [5]) mediated by GPR43 (FFAR2), they may help prevent an overreaction of the immune system to bacterial invasion, buying time for subsequent, more refined immune regulation and reducing early immunopathological damage.Butyrate (HDACi-mediated) may predominantly play a later “remodeling” and “pro-resolution” role.

By altering gene expression profiles through its HDACi activity (the importance of which in the context of lung diseases is emphasized in [93]), its mechanisms include inhibiting LPS-induced NF-κB activation and the production of pro-inflammatory cytokines TNF-α and IL-1β [80], or regulating NF-κB p65 via HDAC1 inhibition in asthma models [81], and potentially directly inhibiting the TLR2/MyD88/NF-κB pathway to ameliorate LPS-induced ALI [90]. Concurrently, butyrate may also influence macrophage polarization towards the M2 type (although the effect is context-dependent [83]), enhance anti-inflammatory signals (including IL-10, promoting Treg differentiation [91]), and promote tissue repair-related pathways. These actions collectively help drive the transition of inflammation from a destructive phase to resolution and recovery phases.

In vivo experiments also indirectly show that increased overall SCFA levels are associated with improved pulmonary inflammation.

However, the regulation of TLR2 signaling by butyrate through HDAC inhibition is not always inhibitory. As discussed above, there is evidence that the HDACi effect of butyrate may synergistically enhance certain specific downstream branches of the TLR2 signaling pathway. One study showed that in THP-1 cells, butyrate, through HDAC inhibition, can enhance NLRP3 inflammasome activation and IL-1β secretion induced by the TLR2 ligand LTA [94].

Additionally, in keratinocytes, HDACi (including butyrate) synergize with vitamin D3 to enhance the expression of the antimicrobial peptide cathelicidin through a TLR2-dependent mechanism [92]. These findings suggest that in the later stages of the inflammatory response, butyrate may not only "inhibit" or "promote resolution" but more likely plays the role of a “fine-tuner”: it may, while suppressing excessive and detrimental inflammatory responses (e.g., via the NF-κB pathway), selectively enhance certain TLR2 downstream effects that are crucial for host defense and restoration of tissue homeostasis (e.g., inflammasome responses to specific pathogens or antimicrobial peptide production). The complexity and context-dependency of this effect (depending on cell type, stimulus, microenvironment, etc.) are key questions that require in-depth exploration in future research.

This hypothesized differential regulatory model—wherein acetate/propionate provide early inhibition, while butyrate regulates late-stage inflammation resolution and may synergistically enhance specific defense responses—if confirmed, would offer new perspectives on understanding how gut microbiota metabolites finely regulate the complex immune response of the lungs to infection via the gut–lung axis.

Knowledge gaps and challenges

Despite some progress in research, numerous critical knowledge gaps and challenges still exist in the field of SCFA–TLR2 axis research:The most critical gap: although existing research [5] suggests a direct antagonistic effect of FFAR2 on TLR2 signaling (by inhibiting the TAK1-NF-κB axis in lung cancer cell lines), and another study [90] links the protective effect of butyrate to the inhibition of the TLR2/MyD88/NF-κB pathway (in an LPS-induced ALI model), there is still an extreme lack of direct and systematic experimental evidence to definitively confirm and differentiate the direct regulatory effects of acetate, propionate, and butyrate on the TLR2 signaling pathway (including receptor expression, activation of all key signaling molecules, and complete downstream gene expression profiles) in primary pulmonary immune cells (especially alveolar macrophages and epithelial cells) and their differences. This is a core bottleneck in understanding the function of this axis.Lack of direct in vivo comparative studies: there is currently a lack of systematic in vivo comparative studies in bacterial pneumonia models that directly compare the effects of these three major SCFAs (administered via different routes and dosages) on the activation state of the pulmonary TLR2 pathway, downstream inflammatory responses (including pro-/anti-inflammatory cytokine profiles, immune cell phenotypes and functions), pathogen clearance efficiency, and pulmonary pathological changes.In-depth elucidation of precise molecular mechanisms and crosstalk: the precise molecular mechanisms of action for different SCFAs, particularly whether complex crosstalk exists between GPR signaling pathways and HDAC inhibition pathways in specific pulmonary cells, and how they collectively or antagonistically fine-tune specific branches of the TLR2 signaling network (including NF-κB, MAPKs, IRFs, inflammasomes), along with the molecular details and functional significance of such crosstalk, remain incompletely understood. Some studies suggest that the HDACi effect of butyrate may synergistically enhance certain downstream responses of TLR2, which contrasts with the widely perceived inhibitory role.

More in-depth research is urgently needed to clarify the sophisticated regulatory logic behind these seemingly contradictory phenomena [92, 94].Context-dependency of effects: the regulatory effects of SCFAs on TLR2 signaling are likely to be strongly influenced by multiple factors, including different stages of infection (early versus late), different pathogens (such as Streptococcus pneumoniae versus Staphylococcus aureus, or viral/fungal co-infections, considering TLR2 recognizes all these pathogens), the host’s baseline status (such as age, sex, genetic background, presence of underlying diseases like COPD/diabetes, which themselves can affect TLR2 expression and function [32, 68]), and the composition and activity state of the gut microbiota itself. These complex context-dependent differences await systematic investigation.Clinical translation challenges: even if more convincing evidence is obtained in animal models in the future, translating these findings safely and effectively into clinical intervention strategies for human bacterial pneumonia still faces enormous challenges. This includes determining the optimal administration routes for SCFAs or their derivatives (oral, inhalation, targeted delivery), effective and safe dosage ranges, accurately assessing inter-individual differences in response to interventions (especially in critically ill patients), and thoroughly considering potential side effects (e.g., excessive inhibition of early TLR2 signaling might impair pathogen clearance, while over-enhancement of certain inflammatory branches could exacerbate immunopathology).

Future research directions

To fill the aforementioned knowledge gaps and promote substantial progress in this field, future research endeavors urgently need to adopt multi-dimensional, integrative strategies, focusing on the following key directions:Prioritize mechanisms, focusing on direct evidence: priority must be given to employing advanced in vitro co-culture models (including primary human/murine alveolar macrophages, lung epithelial cells, or more complex organoids/air–liquid interface culture models) and rigorous in vivo models (including the use of cell-type-specific GPR41/43/109A or key HDAC subtype gene knockout/overexpression mice, combined with lung-specific administration/infection methods). These should be used to directly compare the direct effects of physiologically and pathologically relevant concentrations of acetate, propionate, and butyrate on the pulmonary cell TLR2 signaling pathway (from receptors to transcription factors to effector molecules), and to thoroughly analyze their molecular details (investigating which specific HDAC subtypes are involved; whether direct protein interactions or signal integration points exist between GPR and HDACi pathways).Embrace omics, map the panoramic landscape: high-throughput omics technologies should be widely applied, including single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, proteomics, metabolomics, and epigenomics (such as ChIP-seq, ATAC-seq, to study specific histone modifications and chromatin accessibility changes). These should be used to comprehensively and unbiasedly reveal the overall regulatory landscape, dynamic changes, and cellular heterogeneity of the effects of different SCFAs at various time points on the pulmonary immune microenvironment (including various immune and structural cells) and TLR2-related signaling networks.Simulate clinical scenarios, enhance translational value: there is a need to systematically validate the role of the SCFA–TLR2 axis and its impact on disease outcomes (including pathogen clearance, extent of lung injury, inflammation resolution kinetics, long-term complications such as fibrosis) in animal models of bacterial pneumonia that more closely mimic clinical reality.

Specific factors to consider include aged animals, animals with comorbidities like COPD or diabetes, different sexes, models infected with different clinically common and TLR2-relevant pathogens like Streptococcus pneumoniae and MRSA, and models with varying infection doses and disease severities. This will enhance the clinical extrapolation potential and translational value of research findings.Precision intervention, explore therapeutic potential: based on a deeper understanding of the mechanisms, more specific and precise intervention strategies can be explored in the future. This may include developing highly selective GPR agonists/antagonists, inhibitors of specific HDAC subtypes (to reduce potential side effects of pan-HDAC inhibition), or designing precision microbiome-based therapies (such as specific prebiotic combinations, engineered bacteria, optimization of fecal microbiota transplantation) that can target and regulate the gut microbiota to optimize the production and ratio of endogenous SCFAs (especially butyrate).

The potential of these approaches as adjunctive therapies for bacterial pneumonia should also be assessed.Connect to clinical practice, validate human relevance: well-designed human observational studies (such as prospective cohort studies) should be encouraged. These studies should analyze the association between SCFA levels in pneumonia patients (with different pathogen infections and varying severities) (in feces, blood, or even BALF) and TLR2 pathway-related biomarkers, clinical phenotypes, disease progression, and prognosis. After obtaining sufficient and reliable preclinical evidence, proof-of-concept clinical intervention trials, with thorough assessment of safety and efficacy, should be cautiously designed and conducted to ultimately determine the practical value of SCFA-related interventions in the prevention and treatment of human bacterial pneumonia.

Through the multi-faceted and in-depth research described above, it is hoped that the complex mechanisms by which gut-derived SCFAs participate in the inflammatory balance of bacterial pneumonia by regulating the pulmonary TLR2 signaling pathway will be gradually unveiled. This will not only deepen our understanding of gut–lung axis interactions, but may also provide a solid scientific basis for developing novel microbiome-based strategies for the prevention and treatment of pneumonia.

Conclusion and outlook

This review highlights the critical role of the SCFA–TLR2 axis in orchestrating the delicate inflammatory balance during bacterial pneumonia. By synthesizing current mechanistic understanding, we propose a differential regulatory model: acetate and propionate likely attenuate early inflammatory overactivation primarily via rapid GPR-mediated signaling, whereas butyrate facilitates late-stage inflammation resolution and tissue repair through epigenetic remodeling via HDAC inhibition. This framework offers a novel perspective on how the gut microbiota finely tunes pulmonary immune responses.

However, substantial knowledge gaps remain. The field is currently limited by a scarcity of direct experimental evidence comparing these distinct mechanisms in primary pulmonary cells and clinically relevant in vivo models. Future research must prioritize integrating high-throughput omics technologies with cell-type-specific genetic models to decipher the precise molecular crosstalk between GPR and HDAC pathways.

Addressing these challenges will be pivotal in translating gut–lung axis interactions into safe and effective precision microbiome-based therapies for bacterial pneumonia.