Interactions between the gut microbiota and immune cell dynamics: novel insights into the gut-bone axis

ABSTRACT

Over the past few decades, accumulating evidence has demonstrated that gut microbiota engages in a sustained dialog with the immune system, leading to microbiota-driven immune responses that mediate the regulation of bone-related diseases. Despite the complexity of the dynamic interactions within the gut-immune-bone axis, advancements in high-throughput multi-omics sequencing have significantly facilitated the detailed exploration of this intricate network, thereby providing the potential to develop novel therapeutic strategies for bone-related diseases. In this review, we first summarize the variations in gut microbiota composition observed in patients with bone-related diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), and osteoporosis (OP), in comparison to healthy controls, along with the factors influencing these changes. The review that follows synthesize evidences highlighting the profound effects of gut microbial dysbiosis on immune homeostasis and bone microenvironment, respectively. We further elaborate that the gut-immune axis and gut-bone axis are not independent but three-dimensional networks, emphasizing gut microbial dysbiosis as a pivotal driver of immune dysregulation and subsequent bone homeostasis imbalance. Therapeutic strategies to manipulate the gut-immune-bone axis based on the use of probiotics as well as prebiotics, fecal microbiota transplantation, dietary modifications, and pharmacological interventions are also discussed. Finally, we discuss the challenges of current research on the gut-immune-bone axis and propose future directions for identifying novel therapeutic targets based on this axis to treat these diseases.

Introduction

With the global population aging at an unprecedented rate, bone-related diseases have emerged as health concerns that impose substantial socioeconomic burdens worldwide.¹ Osteoporosis (OP), osteoarthritis (OA), and rheumatoid arthritis (RA) collectively constitute a high incidence of bone-related diseases plaguing middle-aged and elderly populations.^2–4 These diseases share a fundamental pathogenic mechanism: the disruption of bone homeostasis. Bone is a dynamic and metabolically active tissue that undergoes continuous remodeling throughout life, a process that is tightly regulated by the balanced activities of osteoclasts (OCs) and osteoblasts (OBs).⁵ The metabolic balance of bone tissue is paramount for skeletal integrity and functionality. Nevertheless, bone homeostasis may be compromised by a range of endogenous and exogenous factors that may exacerbate OCs activity or attenuate OBs differentiation capacity within the bone microenvironment, ultimately leading to an imbalance in bone homeostasis.^6,7 Furthermore, age-related skeletal decline, which begins as early as the third decade of life, manifests as a progressive loss of bone mass and deterioration of bone microstructure.⁸ Currently, the precise mechanisms underlying bone-related diseases are not fully elucidated, and existing therapeutic strategies primarily alleviate symptoms rather than providing definitive cures. Therefore, there is a pressing need to advance research into bone-related diseases to elucidate the underlying molecular and cellular mechanisms.

Gut microbiota is increasingly recognized as a crucial factor in regulating host health. It is responsible for regulating nutrient metabolism, energy harvest, mucosal immunity, systemic immune response, and neurological development.⁹ Emerging evidence indicates that gut microbiota drives specific gut-organ axes implicated in the pathogenesis of multiple diseases.¹⁰ Among them, the gut-bone axis has been acknowledged as a critical pathway to mediate bone health.¹¹ Studies have demonstrated that dysregulated gut microbiota-derived signals could modulate the systemic inflammatory environment and local bone remodeling through both direct and indirect pathways.¹² These interactions disrupt OBs-OCs crosstalk, ultimately leading to abnormal bone remodeling dynamics and impaired skeletal integrity.¹³ However, with further research, it has been determined that the gut-bone axis constitutes a complex and multilayered regulatory network, rather than a straightforward bidirectional pathway. Within this intricate network, the immune system has gained increasing attention as a key mediator. The field of osteoimmunology, initially conceptualized by Arron and Choi in 2000, systematically investigated the dynamic crosstalk between bone and immune system, which suggests that immune dysregulation could directly disrupt bone homeostasis.¹⁴ Subsequently, in 2018, Rupesh K. Srivastava et al. initially introduced the novel field of “Immunoporosis.” They advocated for in-depth investigations into the mechanisms of action of various T lymphocyte subsets in OP and further emphasized the significance of developing targeted therapeutic strategies aimed at modulating immune cells to prevent and treat OP.¹⁵ It is noteworthy that the gut microbiota has a profound impact on the host immune system.¹⁶ Based on these insights, we propose a novel gut-immune-bone axis research paradigm, in which gut microbiota dysbiosis may trigger immune dysfunction, thereby altering the balance of bone remodeling and predisposing individuals to bone diseases.

Despite significant advancements in the exploration of the gut-bone axis, systematic reviews comprehensively investigating the mediated role of the immune system within the gut-bone axis remain notably absent from current literature. This review aims to elucidate specific pathways of the gut-immune-bone axis, with a particular emphasis on the dynamic interactions between gut microbiota, immunomodulation, and bone homeostasis. We further evaluated the therapeutic potential of targeting this axis through microbiota-targeting interventions, including probiotics, fecal microbiota transplantation (FMT), dietary, and pharmacological approaches. The paradigm of the gut-immune-bone axis presented herein represents a significant conceptual advance with important implications for both basic research and clinical management of bone-related diseases.

Gut microbial dysbiosis in bone-related diseases

The gut microbiota, constituting the most extensive microecosystem within the human body, is essential for maintaining bone health through the intricate mechanism of the gut-bone axis.¹¹ In comparison to healthy individuals, patients with RA, OA, or OP exhibit significant differences in the function and composition of gut microbiota, which are closely associated with disease progression Table 1.³¹ Of particular significance is the fact that multiple factors such as diet, age, and gender could influence bone homeostasis by regulating gut microbiota, thereby contributing to the initiation and progression of bone-related diseases Figure 1.^32–35

Table 1.

Alterations of gut microbiota in bone-related diseases.

Diseases	Gut microbiota			References
	phylum	Upregulation	Downregulation
Rheumatoid arthritis	Firmicutes	Lactobacillus Prevotella Streptococcus Veillonella parvula Lachnospiraceae	Collinsella Romboutsia Ruminococcus_2 Coprococcus_2 Ruminococcaceae_UCG-009	41,17,40,18
	Bacteroidetes	Bacteroides uniformis Prevotella copri Eubacterium rectale Prevotella Bacteroides plebeius	Faecalibacterium prausnitzii Bacteroidaceae Parabacteroides distasonis Alloprevotella Rikenellaceae RC9	19,41,37,20,35
	Actinobacteria	Bifidobacterium dentium Lachnospiraceae	Bifidobacterium Collinsella Bifidobacterium longum Coriobacteriaceae Erysipelotrichaceae	41,21,35
	Proteobacteria	Neisseria Pseudomonas Escherichia-Shigella	Pseudomonas Enterobacter Klebsiella Citrobacter	40,35
Osteoarthritis	Firmicutes	Streptococcus Blautia Christensenellaceae Lactobacillus H mucosae Peptococcaceae	Phascolarctobacterium Roseburia Lachnospiraceae Roseburia intestinalis Anaeroflum	22,61,23,59
	Bacteroidetes	Desulfovibrio Bilophila wadsworthia Bilophila Desulfovibrionaceae Hungatella hathewayi	Bacteroides spp	61,23
	Actinobacteria	Propionibacterium	Bifidobacterium	22,24
	Proteobacteria	Citrobacter B koseri Kingella spp. Shimwellia Intestinimonas	Haemophilus spp. Citrobacter spp.	25,61,23
Osteoporosis	Firmicutes	Lactobacillus Enterobacteriaceae Veillonella	Bacteroides fragilis Faecalibacteriums Ruminococcaceae Blautia Ruminococcus	26,27,147,28,29
	Bacteroidetes	Faecalibacterium Parabacteroides Eggerthella Prevotellaceae_UGG-001 Harryflintia	Bacteroides Prevotella Prevotella	24,26,30,147,28
	Actinobacteria	Actinomycetaceae Streptococcaceae	N/A	27
	Proteobacteria	Gammaproteobacteria Escherichia Shigella Klebsiella	N/A	24

Figure 1.

Association between gut dysbiosis and bone-related diseases.

Patients with bone-related diseases such as RA, OA, and OP exhibit significant alterations in both composition and function of the gut microbiota, suggesting a critical role of gut microbiota in the pathogenesis of these diseases. Furthermore, gut dysbiosis arises from multiple contributing factors, particularly dietary patterns, age-related changes, and gender-specific variations. These factors disrupt the equilibrium between beneficial and pathogenic bacterial populations, thereby promoting inflammatory responses and pain manifestations in anatomically distinct regions, including the hands, hips, and knees. Created with BioRender.com

RA

RA is an autoimmune disease characterized by chronic joint inflammation, pain, and joint degeneration. Epidemiological studies indicated that the global prevalence of RA has reached 17.6 million cases in 2020, representing a 121% increase since 1990, with projections suggesting a further rise to 31.7 million cases by 2050. The gut microbiota, acknowledged as a critical regulator, is progressively drawing attention for its potential involvement in the pathophysiology of RA. It has demonstrated that the interaction between gut microbiota and the immune system significantly influences susceptibility to type 1 diabetes,³⁶ and a similar mechanism was believed to be operative in RA. Recent studies mapping the gut microbiota of RA patients have identified a significant reduction in microbial diversity compared to healthy controls. This alteration is characterized by an increased presence of pathogenic species, including Prevotella copri as well as Fusobacterium nucleatum, alongside a decreased prevalence of beneficial bacteria, such as Faecalibacterium prausnitzii.³⁷ This gut microbiota dysbiosis could potentially be attributed to the abnormal activation of the immune system in patients with RA. This activation elicited inflammatory responses that eventually disrupt the homeostasis within the gut, giving rise to a decrease in probiotic bacteria or the translocation of harmful bacteria.³⁸ Furthermore, some research suggested that genetic susceptibility genes in RA patients, such as HLA-DR, may indirectly exacerbate the dysregulation of the gut microbiota by modulating immune responses.³⁹ Indeed, such changes were observable even in the preclinical phase, characterized by an increased abundance of Prevotella as well as Blautia gnavus, and a concurrent reduction in Collinsella levels.^18,19,21 Furthermore, transplantation of microbiota from pre-RA patients into mice enhanced collagen-induced arthritis (CIA), a process that was mechanistically associated with disruption of the intestinal barrier and increased T helper (Th) 17 cell infiltration.⁴⁰ The gut microbiota underwent a dynamic and heterogeneous evolution during the progression of RA. Notably, Collinsella aerofaciens became more prevalent during the initial inflammatory stage, whereas Bacteroides uniformis decreased with the onset of cartilage damage. The bone erosion stage was characterized by an increase in Veillonella parvula and Escherichia coli, whereas the late stage was distinguished by an elevated abundance of Prevotella copri. These microbiota fluctuations may be related to impaired intestinal barrier function, inflammatory processes, and disturbances in bone homeostasis.⁴¹ Specifically, Collinsella aerofaciens could contribute to the etiology of RA through the mechanism of protein deamination, producing citrullinated autoantigen peptides that induce the production of anti-citrullinated protein antibodies (ACPA).^20,42,43 Significant heterogeneous changes were observed in the gut microbiota of RA patients, particularly concerning rheumatoid factor (RF) levels. For instance, patients with high RF levels demonstrated a notable reduction in the abundance of Actinobacteria and Firmicutes.⁴⁴ These findings highlight the pronounced heterogeneity of the gut microbiota in RA patients.

Furthermore, diet, medicine, and age are recognized as crucial factors affecting the onset and progression of RA, primarily through their effects on the balance of gut microbiota, and thus the disease trajectory. Previous research has indicated that a high-fiber diet (HFD) could alter the gut microbiota composition, resulting in an increased abundance of Akkermansiaceae and a decreased abundance of Bacteroidaceae. Additionally, the synergistic effect of HFD with Prevotella copri has been associated with exacerbation of RA symptoms.⁴⁵ An interesting observation indicated that alterations in meal timing may also affect the inflammatory cycle of RA by regulating the circadian rhythms of gut microbiota. The abundance of Parabacteroides distasonis demonstrated significant variability between the peak and trough phases of inflammation. This bacterium contributed to the attenuation of RA inflammation through glycine release, a process mediated by the sirtuin 5-nuclear factor-kappa B (NF-κB) axis.⁴⁶

Disease-modifying anti-rheumatic drugs (DMARDs), encompassing both conventional synthetic DMARDs (csDMARDs) and biologic DMARDs (bDMARDs), represent the primary therapeutic agents for RA patients. However, the therapeutic efficacy of these drugs in RA patients could be modulated by interactions with the gut microbiota. For instance, methotrexate (MTX), the most frequently prescribed DMARD for RA treatment, was known to reduce the abundance of Bacteroidetes and increase the abundance of Actinobacteria, thereby alleviating inflammation. Nevertheless, MTX treatment may also induce a reduction in Bacteroides fragilis, leading to gastrointestinal toxicity and other adverse effects.^46,47 In contrast, bDMARDs were primarily employed as alternative therapeutic options for RA patients who exhibit suboptimal responses to conventional DMARDs.⁴⁸ Tumor necrosis factor-alpha (TNF-α) inhibitors, such as etanercept, have been shown to increase the abundance of Cyanobacteria while decreasing the abundance of Proteobacteria, thereby alleviating RA symptoms.⁴⁹ Traditional Chinese medicine has been integral to the treatment of RA for centuries, recognized for its significant efficacy and relatively minimal side effects.⁵⁰ Tripterygium hypoglaucum (Levl.) Hutch (THH) has been shown to enhance the proliferation of beneficial bacterial species such as Bifidobacterium, Akkermansia, and Lactobacillus, while simultaneously reducing the prevalence of detrimental bacteria including Veillonella, Bacteroides, and Anaerostipes, thereby alleviating joint inflammation.⁵¹

Besides, age is also a factor that could influence the composition of the gut microbiota in RA patients. Compared to older RA patients and healthy individuals, young RA patients (<45 years) demonstrated significantly lower gut microbiota diversity, with higher abundances of the Ruminococcus gnavus and the Intestinibacter genus.¹⁷ This phenomenon may be attributed to the fact that the gut microbiota of younger individuals is more susceptible to the influences of disease and environmental factors, leading to an imbalance in the gut microbiota. Moreover, environmental alterations are also a crucial consideration in maintaining gut microbiota homeostasis. Elevated humidity and cold exposure could also exacerbate RA by altering the abundance and metabolic functions of the gut microbiota.^52,53

OA

The Global Burden of Disease for Early-Onset OA study has shown that 98% of countries worldwide experienced a sustained increase in the burden of early-onset OA between 1990 and 2019. By 2019, the number of early-onset OA cases exceeded 21.69 million, the overall prevalence exceeds 140 million, and the total number of YLD exceeds 4.96 million.⁵⁴ Given the increasing prevalence and impact of OA, it is imperative to prioritize its prevention and treatment. Although OA has traditionally been regarded as a degenerative disease, recent studies have reclassified OA as a low-grade inflammatory disorder.⁵⁵ And a growing number of studies have reported that the occurrence of low-grade inflammation is closely related to gut microbiota dysbiosis.

Huang et al. have proposed a “double-hit” model to elucidate the impact of the gut microbiota on OA pathogenesis and exacerbation. This model suggested that gut microbiota could abnormally activate the innate immune system, resulting in the upregulation of systemic inflammatory markers, while also causing direct damage to the joints.⁵⁶ Mendelian randomization analysis of extensive genome-wide association study data has substantiated a causal relationship between specific gut microbiota and the risk of OA. Notably, the abundance of Ruminiclostridium 5 was negatively correlated with the susceptibility to knee OA.⁵⁷ Further research on the gut microbiota composition of OA patients revealed that increased abundance of Actinomycetaceae and Actinomycetales was associated with a heightened risk of developing OA. In contrast, higher prevalence of Desulfovibrio and Parasutterella was correlated with a reduced risk of the disease.⁵⁸ OA predominantly affects weight-bearing joints, such as the knees, hips, and spine. Interestingly, the gut microbiota of OA patients with different onset joints exhibited different changes.³³ Patients with hand OA showed an increased presence of Bilophila and Desulfovibrio, accompanied by a decreased abundance of Roseburia.²³ For patients with knee OA, the gut microbiota was characterized by an increased abundance of Actinobacteria and a decreased abundance of Proteobacteria, with an elevated abundance of Blautia being linked to the development of osteophytes.⁵⁹ Of particular interest is the association between pain symptoms and impaired tryptophan metabolism observed in patients with hand OA. In particular, elevated levels of the tryptophan metabolite 5-hydroxytryptophan processed by the gut microbiota, as well as increased activity of the kynurenine pathway, lead to the accumulation of pain-related metabolites such as kynurenine and kynurenine aldehyde. These metabolites contribute to the onset of pain symptoms in patients with hand OA.^60,61 These findings suggest that alterations in the composition and metabolic activity of the gut microbiota may play a key role in the pathogenesis of OA.

The clinical management of OA is challenging due to its multifactorial etiology, including factors such as gender differences, obesity, and joint injury.⁶² It is plausible that these factors may facilitate the onset of OA through their impact on gut microbiota regulation. OA patients frequently present comorbidities of obesity and insulin resistance. Dysregulated lipid metabolism and high-fat dietary patterns are associated with a decline in beneficial bacteria, such as Bifidobacterium, and an overgrowth of opportunistic pathogens, including Enterobacteriaceae. Moreover, the majority of OA patients are elderly individuals. As age advances, intestinal barrier integrity diminishes, accompanied by elevated levels of oxidative stress. These factors may impede the establishment and maintenance of beneficial bacterial populations within the intestinal tract. Empirical evidence from antibiotic intervention studies has highlighted the potential of modulating gut microbiota to mitigate bone sclerosis and cartilage degeneration in OA mice model.⁶³ It is worth noting that female patients with OA experienced more severe pain than male patients at the same condition grade.⁶⁴ Correspondingly, transplantation of the gut microbiota from female mice to male mice alleviated OA symptoms, whereas reverse transplantation exacerbated the condition, confirming the distinct influence of the gut microbiota on sex-based OA outcomes.⁶⁵ Further studies of OA found that obesity-related OA is not only a consequence of mechanical overload but is also driven by the combined effects of metabolic disturbances and chronic inflammation, which are strongly linked to imbalances in the gut microbiota.^22,66 It has been demonstrated that HFD in obese mice is associated with reduced gut microbiota diversity, characterized by a decreased abundance of beneficial microbiota including Bifidobacteria but an increased presence of pro-inflammatory Peptostreptococcus. This alteration resulted in increased intestinal permeability, facilitating the entry of detrimental substances, such as endotoxins, into the bloodstream. Therefore, this triggered systemic inflammatory responses, which in turn exacerbate the progression of OA.²² Furthermore, in the presence of joint damage, there was a reduction in the population of anti-inflammatory Bacteroidetes and a corresponding increase in pro-inflammatory Firmicutes, potentially exacerbating OA progression through the induction of a persistent low-grade inflammatory state.⁶⁷ The findings are consistent with the “two-hit model” hypothesis, which proposes that gut microbiota dysbiosis serves as the initial trigger by activating the innate immune system (the first hit), followed by joint injury (the second hit), thereby orchestrating synergistic pathogenic effects.⁵⁶ Notably, independent associations between gut microbiota dysregulation and cartilage damage remained even when obesity and joint damage were excluded as confounding variables.²⁵ This finding further supports the association between gut microbiota composition and OA severity. Besides, vitamin D intervention showed potential in the early stages of OA, whereas vitamin D deficiency was associated with increased risk of disease progression.⁶⁸ Vitamin D not only facilitated calcium absorption but also participated in maintaining intestinal mucosal homeostasis. In the case of vitamin D deficiency, intestinal mucosal dysfunction induced gut microbiota dysbiosis, thereby reducing calcium absorption and ultimately leading to decreased bone density.³¹ The underlying mechanism may be based on the fact that the gut microbiota of patients with knee OA showed increased abundance of Peptococci and decreased abundance of Delftia, while vitamin D deficiency further exacerbated the aberrant proliferation of Parabacteroides and Butyricimonas, which in turn aggravated intestinal barrier damage and inflammatory response.⁶⁹

OP

Studies have found that the number of OP cases worldwide doubled, with projections suggesting that the number will rise to 263 million between 2030 and 2034. Consequently, addressing the prevention and management of OP is essential to reduce social stress and improve overall quality of life. It has been demonstrated that there is a pronounced correlation between bone mineral density and gut microbiota composition.⁷⁰ However, the underlying mechanism for the coupling between them is intricate.

OP is divided into primary and secondary types based on its etiology and underlying mechanisms. More prevalent primary types include postmenopausal, senile, and idiopathic subtypes and are thus related to factors such as age, sex, and genetic predisposition. The secondary types are triggered by diseases such as endocrine diseases, inflammatory processes, or the use of certain medications.⁷¹ In recent years, the involvement of immune mechanisms in the pathogenesis of osteoporosis has emerged as a significant area of investigation. Immunoporosis, a secondary type of osteoporosis, is marked by immune system dysregulation, in which immune cells and cytokines facilitate osteoclastogenesis and maturation, thereby augmenting bone resorption, while concurrently suppressing OB activity and impeding bone formation, ultimately resulting in the development of OP.⁷² Current studies predominantly focus on the elderly population, where studies have identified an increased abundance of Actinomyces, Eggerthella, Clostridium Cluster XlVa, and Lactobacillus within their gut microbiota. Conversely, the abundance of Escherichia, Shigella, and Weyonella was significantly reduced.³⁰ In the adolescent patient population, a distinct pattern emerged with a reduction in Bacteroidetes and an increase in Firmicutes,²⁶ highlighting the significant effect of age on gut microbiota composition. The majority of studies propose that the dysbiosis of the gut microbiota in OP patients is closely associated with estrogen deficiency. In postmenopausal women, estrogen deficiency not only compromised intestinal barrier integrity but also reduced the abundance of calcium-absorbing bacteria, particularly Lactobacillus species.³¹ The estrogen-dependent gut dysbiosis in turn disrupted mineral homeostasis and significantly elevated the risk of developing postmenopausal osteoporosis (PMO). Wang et al. have revealed reduction in both α and β diversity within the gut microbiota of PMO patients.⁷³ In contrast, Yan et al. have indicated an increase in the richness and diversity of gut microbiota in PMO patients, characterized by a simultaneous increase in Firmicutes and Lactobacillus populations but significant decreases in Bacteroides.²⁸ However, PMO patients demonstrated downregulation of the intestinal tryptophan metabolic pathway, resulting in significantly lower levels of their metabolites, such as indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA), accompanied by higher serum lipopolysaccharide (LPS) concentrations, thereby exacerbating bone loss. These alterations were able to be reversed through antibiotic therapy.^74,75 It is worth noting that irrespective of whether OP is induced by estrogen deficiency, immune cells such as T cells and macrophages can modulate bone metabolism through the conventional receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway, while also regulating bone mass via immunomodulatory mediators and cell-cell interactions.⁷⁶ These findings suggest a strong association between the gut-bone axis and the pathogenesis of OP.

Moreover, variations in gut microbiota composition were observed among different populations with OP. Patients from Nordic regions exhibited a higher abundance of Proteobacteria but a lower abundance of Tenericutes.²⁴ In contrast, the gut microbiota of Chinese patients was characterized by an increase in Actinomyces and Streptococcus, along with a reduction in Akkermansia, Bacteroides, and Butyricimonas synergistica.²⁷ The differences in regional diet, culture, and climate may explain the significant variations of gut microbiota in patients with OP among different populations. In similar circumstances, dietary patterns are instrumental in modulating the gut-metabolite-bone axis. Western dietary patterns characterized by high fat as well as sugar and low fiber have been shown to induce osteopenia in mice, accompanied by the accumulation of bone marrow fat and inhibition of bone formation. This effect was associated with alterations in the gut microbiota composition, specifically an increase in Firmicutes and a decrease in Bacteroidetes, Actinobacteria, and Proteobacteria. Furthermore, the high-fat component of the diet exacerbated bone loss.⁷⁷ Eastern dietary patterns were observed to promote gut microbiota diversity compared to Western diets, thus potentially reducing the risk of OP.⁷⁸ Additionally, the composition and function of the gut microbiota in patients with OP are modulated by pharmacological interventions and environmental factors. Although glucocorticoids (GCs) are effective in treatment for immune-related disorders, their use has been associated with the onset of OP and, in severe cases, osteonecrosis of the femoral head.⁷⁹ GCs therapy resulted in a decrease in the abundance of beneficial Firmicutes and an increase in the abundance of detrimental Bacteroidetes within the gut microbiota of patients. This treatment also undermined the integrity of the intestinal barrier, facilitating the translocation of LPS into the bloodstream, which subsequently contributes to the onset of OP.⁸⁰ Furthermore, another study showed that GCs exacerbated intestinal inflammation by reducing the abundance of Lachnospirillaceae and subsequently suppressing butyrate production.⁸¹ In addition, the prevalence of lower bone mineral density among high-altitude inhabitants appeared to be associated with an increased prevalence of Catenibacterium, particularly in male and elderly populations.²⁹ These findings suggest regional differences in gut microbiota composition in patients with OP.

In conclusion, the pathogenesis of bone-related diseases is closely associated with gut microbiota dysbiosis, which is influenced by multiple factors including age, gender, and diet. As studies on the commonalities of gut microbiota alterations in these three diseases progress, the critical differences in microbial research among them tend to be neglected. The distinct research models used for the three diseases dictate the direction of gut microbiota alterations. Research on gut microbiota in RA patients mainly utilized the CIA model to mimic autoimmune dysregulation. For OA patients, gut microbiota research typically relied on high-fat diet or surgical induction models to replicate the combined effects of metabolic inflammation and mechanical stress. In the case of OP patients, the OVX model is frequently employed to mimic the imbalance between bone formation and bone resorption. Furthermore, the limited comprehension of gut microbiota dynamics and the disparities in the application of gut microbiota sequencing techniques represent major challenges in microbial research. A more comprehensive understanding of the differences in microbial research across these three diseases will facilitate the elucidation of their complex pathogenic mechanisms. To overcome these key challenges, future studies should prioritize the development of a global human gut microbiota database that integrates individual factors, thereby enabling a more accurate assessment of gut microbiota composition across diverse populations. Simultaneously, the optimization of high-throughput sequencing technologies to improve accuracy and depth will enable the identification of microbial patterns associated with disease progression. Additionally, standardizing microbial research protocols to enhance the consistency and reliability of results will contribute to a deeper understanding of the pathogenic mechanisms of the gut microbiota in individuals with the same disease.

Gut microbiota-driven modulation of the immune system: mechanisms and consequences

The interaction mechanisms between the gut microbiota and the immune system have emerged as a prominent research focus in recent years, with ongoing results highlighting their links to human health and disease progression.⁸² The gut is not only the site of material metabolism but also the central physiological nexus responsible for essential functions such as information transmission and metabolic regulation.⁸³ This positions it as a crucial player in maintaining intricate interactions with various systemic components of the body. Due to its direct exposure to the external environment, the gut has developed a distinctive immune defense system that ensures host health by maintaining microbial homeostasis and immune balance, a regulatory mechanism known as the gut-immune axis.⁸² Disruptions in the composition of the gut microbiota or abnormal metabolism could trigger immune cell dysfunction and compromise the integrity of the intestinal barrier Figure 2. These disturbances, along with systemic inflammatory responses, could ultimately contribute to the emergence of a wide range of pathological conditions Table 2.

Figure 2.

Immunomodulatory mechanisms of gut microbiota.

The gut microbiota regulates the differentiation and functional activation of diverse immune cell populations through microbial-derived metabolites, thereby maintaining immune homeostasis. Furthermore, these microbial communities orchestrate the systemic migration of immune cells via circulatory pathways to peripheral organs, thereby establishing bidirectional communication between gut microbiota and extra-intestinal organ systems. Created with BioRender.com

Table 2.

Modulation of the immune system by the gut microbiota.

Immunomodulatory Pathway	Immune cells	Metabolites of the gut microbiota	Mechanism	References
Immune cell differentiation balance	Treg/Th17	SCFAs	Promote the differentiation of Treg cells and suppress the differentiation of Th17 cells	84–91
		Secondary bile acid		92,93
		Tryptophan metabolites		91,94
	M1/M2	SCFAs	Inhibit M1 polarization and promote M2 polarization,	95–97
		Tryptophan metabolites		98,99
		Putrescine	Promote M2 polarization	145
		3,4-DHPPA	Inhibit M1 polarization	100
		TMAO	Promote M1 polarization	146
		Succinic acid		147
Immune cell trafficking	TNF+ T cell	SFB	Promote the migration of TNF+ T cells from the intestine to the bone	101,126
	Th17 cell	SFB	Promote the migration of Th17 cells from the intestine to the bone	101,126
		Tryptophan metabolites		74
	Macrophages	LPS	Promote the migration of macrophages from the intestine to the brain	102
	TCRαβ+ T cell	SCFA	Promote the migration of TCRαβ+ T cells from the intestine to the brain	103,104,150
	Treg cell	SCFAs	Promote the migration of Treg cells to the pancreas	105
	ILC3s	WMT	Promote the migration of ILC3s to the liver	106
Regulation innate immune cells response	ILCs	SCFAs	Inhibiting GATA3 expression in ILC2	107
			Promote the generation of IL-22 by ILC3	108,109
		PA	Promote the release of IL-13 by ILC2	110
		Secondary bile acid	Promote the differentiation of ILC1	111
		Tryptophan metabolites	Inhibit the generation of IL-22 by ILC3	162
			Promote ILC3 to restrain the generation of IL-17	163,164
	DCs	SCFAs	Regulate the functions and recruitment of DCs	112,113
		Secondary bile acid	Inhibition of Th17 cells and promotion of Treg cells via DC to alleviate inflammation	114–117
		L-lysine	Inhibition of IL-17 secretion by Th17 cells through DC	118
		PSA	Promote the secretion of IL-10 from Treg cells and suppress inflammation	119
Regulation adaptive immune cells response	B cell	LPS	Induce the abnormal proliferation of IgA+ plasma cells and trigger mucosal immune disorder	59,120
		SCFAs	Promote Breg cells differentiation	121
		Tryptophan metabolites	Promote Breg cells differentiation	122
	Tfh cell	SFB	Promote the differentiation of Tfh cells	123

Immune cell subset differentiation

Regulatory T (Treg) cells/Th17 cells

Th17 and Treg cells are subsets of CD4⁺ T cells, and the Treg/Th17 balance formed by their mutual antagonism plays a crucial role in the occurrence and development of certain inflammatory diseases, including autoimmune diseases.¹²⁴ In animal experiments, single-cell RNA sequencing (scRNA-seq) demonstrated that the depletion of the gut microbiota resulted in a marked decrease in the population of Treg and Th17 cells within the spleen of mice.¹²⁵ Indeed, clinical observations have identified a significant increase in the Th17 cell population in patients at the preclinical stage of RA, suggesting a potential correlation between Th17 cells and the onset of RA.⁴⁰ Furthermore, the expansion of Th17 cells was associated with OP progression, with emerging evidence indicating that the resultant bone loss may be mediated through the modulation of segmented filamentous bacteria.^101,126 This finding underscores the critical role of the gut microbiota in maintaining the Treg/Th17 balance.

Emerging evidence has demonstrated that gut microbiota could modulate Treg/Th17 balance through microbial-derived metabolites, such as short-chain fatty acids (SCFAs), secondary bile acids, and tryptophan metabolites.^127,128 SCFAs, which include acetate, propionate, and butyrate, are produced by the fermentation of dietary fiber by the gut microbiota.¹²⁹ It has been shown that these SCFAs could promote the differentiation of Treg cells by inhibiting histone deacetylase (HDAC) activity and enhancing forkhead box P (Foxp) 3 expression.^84,91 Specifically, butyrate could not only induce the acetylation of estrogen-related receptor alpha by inhibiting HDAC8, regulate the expression of carnitine palmitoyltransferase 1 and nuclear receptor subfamily 1 group D member 1, and increase the amount of Treg cells,⁸⁶ but also restore the Treg/Th17 balance by activating the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma (PPARγ) pathway and inhibiting signal transducer and activator of transcription (STAT) 3 phosphorylation, thereby alleviating colitis.^88,89 Furthermore, acetate produced by Blautia has been shown to reduce the proportion of Th17 cells but increase the proportion of Treg cells by activating the G-protein coupled receptor (GPR) 43. Concurrently, it could inhibit the release of pro-inflammatory cytokines, thereby alleviating inflammatory responses.⁹⁰ In contrast, decreased propionate levels resulting from gut microbiota dysbiosis decreased its ability to activate GPR43 and inhibit HDAC6, which subsequently inhibited Treg differentiation and promoted Th17 differentiation.⁸⁵ Of particular significance is the bidirectional regulation between SCFAs and Treg cells. SCFAs activated free fatty acid receptor (FFAR) 4 to inhibit the differentiation of Th17 cells while promoting the differentiation of Treg cells.⁸⁷ In turn, Treg cells contributed to the maintenance of SCFA levels through the restoration of FFAR2/FFAR3 expression.¹³⁰ It is worth noting that most secondary bile acid metabolites produced by the gut microbiota could likewise exert regulatory effects on the Treg/Th17 balance. Studies have shown that 3-oxolithocholic acid and isolithocholic acid could suppress Th17 cell differentiation and facilitate Treg cells differentiation, thereby mitigating inflammatory bowel disease. The underlying mechanism involved activating the vitamin D receptor (VDR) and inhibiting the transcriptional activity of the retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) protein.^131–133 In autoimmune encephalomyelitis, 24-deoxyursodeoxycholic acid reduced the quantity of Th17 cells and inhibited their capacity to produce IL-17A by suppressing the glutamine metabolism that is a prerequisite for mechanistic target of rapamycin complex 1 activation and glycolysis, thus promoting the generation of Treg cells and exerting anti-inflammatory actions.¹³⁴ Similarly, deoxycholic acid (DCA) and lithocholic acid (LCA) have analogous functions.¹³⁵ Furthermore, Lactobacillus murinus (L. murinus) was capable of elevating the levels of tryptophan metabolites indole acrylic acid (IA) and indole-3-aldehyde (IAld) in the gut microbiota, thereby regulating the Treg/Th17 balance through the activation of aryl hydrocarbon receptor (AhR) and alleviating pulmonary inflammation.⁹¹

M1/M2

Since the introduction of the “phagocyte” concept by Metchnikoff, macrophages have evolved from being perceived merely as pathogen defenders to being recognized as key regulators of microenvironmental stability. Recent research suggested that macrophages, particularly due to their unique phenotypic adaptability, function as “biological transducers,” converting microenvironmental signals into diverse immune responses. This dynamic adjustment mechanism was essential in maintaining tissue homeostasis and regulating pathological processes.¹³⁶ The functional versatility of macrophages is exemplified in the dynamic balance between the classical activation (M1) and alternative activation (M2) polarization states. M1 macrophages are involved in immune defense by releasing pro-inflammatory factors such as TNF-α and IL-6, whereas M2 macrophages contribute to anti-inflammatory responses, tissue repair, and remodeling through mediators like transforming growth factor-beta (TGF-β) and IL-10.¹³⁷ Notably, these polarization states do not represent fixed phenotypes but rather a continuous functional spectrum, with their dynamic equilibrium being finely modulated by the gut microbiota.¹³⁸ Emerging evidence suggested that the gut microbiota exerts profound effects on the tumor immune microenvironment through the reprogramming of transcriptional networks within immune cells, consequently influencing the composition and functional dynamics of immune cell populations. George B.H. Green’s team implanted mouse glioma cells into the brains of two humanized mouse models exhibiting distinct gut microbiota compositions: HuM1 (dominated by Alistipes) and HuM2 (dominated by Odoribacter). Following scRNA-seq of tumor-infiltrating immune cells, it was found that HuM1 mice displayed a significantly higher proportion of M1-polarized macrophages relative to HuM2 mice, accompanied by upregulation of pro-inflammatory markers (Nos2) and downregulation of immunosuppressive markers (Arg1 and Mrc1).¹³⁹ Furthermore, Yifeng Lin et al. have demonstrated through analysis of scRNA-seq and in vivo experiments that Bifidobacterium adolescentis could activate the TLR2/YAP signaling pathway, promote macrophage infiltration, upregulate the expression of decorin, and ultimately suppress the progression of colorectal cancer.¹⁴⁰ For bone metabolism, the interconversion of macrophage polarization states exhibits a dual-regulatory function. M1 macrophages promote OCs differentiation via regulating the ratio of RANKL/OPG, while M2 macrophages foster OBs mineralization through the Wingless-related integration site (Wnt)/β-catenin and bone morphogenetic protein (BMP) signaling pathways.^141,142 This dysregulation of macrophage polarization was recognized as the fundamental mechanism responsible for the initiation of bone-related diseases, including OP, OA, and RA.

Recent studies have demonstrated that the gut microbiota, along with its metabolic network, play a crucial role in the regulation of macrophage polarization via the microbiota-immune axis. For instance, F. nucleatum induced M1 macrophage polarization by upregulating protein kinase B (AKT) 2 signaling pathway, thereby exacerbating intestinal inflammatory damage.¹⁴³ Conversely, A. muciniphila facilitated M1 macrophage polarization by stimulating the toll-like receptor (TLR) 2/NF-κB and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathways.¹⁴⁴ However, the gut microbiota exhibits a dual role in macrophage polarization regulation. SCFAs could inhibit M1 macrophage polarization while promoting M2 macrophage polarization, thereby alleviating intestinal inflammatory responses. This regulatory mechanism was associated with the suppression of the mitogen-activated protein kinase (MAPK) signaling pathway within macrophages.^95–97 Furthermore, putrescine has been shown to upregulate protein tyrosine phosphatase non-receptor type 2 expression and inhibit STAT1 phosphorylation, which subsequently promotes M2 polarization.¹⁴⁵ In contrast, trimethylamine N-oxide (TMAO) exacerbated M1 macrophage polarization by activating the NLRP3 inflammasome.¹⁴⁶ Similarly, IAA could demonstrate inhibitory effects on M1 macrophage polarization. Distinctly from previous research, the anti-inflammatory properties of IAA were independent of AhR and instead depended on the activity of heme oxygenase-1.⁹⁸ Ursodeoxycholic acid, a bile acid metabolite derived from the gut microbiota, interacted with the farnesoid X receptor (FXR), leading to the inhibition of NF-κB pathway in macrophages. This interaction facilitated the transition of macrophages from the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype, thereby reducing the production of inflammatory cytokines and promoting tissue repair.⁹⁹ Additionally, within the hepatic environment, the gut microbiota-derived metabolite 3,4-dihydroxyphenylpropionic acid exerted anti-inflammatory effects by inhibiting HDAC, thus preventing M1 macrophage polarization.¹⁰⁰ In the context of lung injury, the gut microbiota metabolite succinate plays a dual role by activating the succinate receptor 1 and the phosphoinositide 3-kinase (PI3K)/AKT/hypoxia-inducible factor 1-alpha signaling pathway, which enhances the polarization of alveolar M1 macrophages and exacerbates lung damage.¹⁴⁷ These findings elucidate the fundamental molecular mechanism by which gut microbiota metabolites function as epigenetic modifiers, orchestrating the functional transition of macrophages through the remodeling of their epigenome.

Immune cell migration

The maintenance of immune homeostasis is contingent upon a sophisticated cellular migration network. As one of the largest immune organs, the mechanism of immune cell migration across tissues in the gut is emerging as a frontier in immunometabolism research.¹⁴⁸ Recent studies have revealed that the intestinal immune system utilizes a “metabolic reprogramming-cellular migration” dual-module regulatory mechanism to establish a dynamic immune network that connects the intestinal mucosal barrier with distal organs. This migratory behavior transcends beyond physical relocation, encompassing multi-level regulatory processes such as chemokine gradient sensing, adaptation to metabolic microenvironments, and epigenetic remodeling.

With the development of cell migration research methodologies, further research has revealed that there are multiple immune cell migration pathways in the gut-bone axis. Zhang et al. employed scRNA-seq to analyze the expression patterns of chemokines Ccr7 and Ccl5 in the spleens of germ-free (GF) and specific pathogen-free (SPF) mice. Their findings demonstrated that the expression of Ccr7 and Ccl5 was markedly downregulated in GF mice compared to SPF controls. Notably, Ccr7 is essential for the regulation of T cell homing, and its downregulation may interfere with T cell migration and compromise the functional integrity of splenic T cells. Ccl5 is crucial for B cell activation and plasma cell differentiation, and its diminished expression may result in compromised humoral immune responses. These results indicated that the gut microbiota dynamically modulates the immune architecture of the spleen by regulating the expression of these chemokines, likely through microbial-derived metabolites that interact with signaling pathways.¹²⁵ Specifically, segmented filamentous bacterium (SFB)-induced Th17 cells and TNF⁺ T cells were transported to the bone marrow via S1PR1-mediated lymphatic homing pathways along the C-C motif chemokine ligand (CCL) 20/CXC chemokine receptor (CXCR) 3 axis. These intestinal-originating Th17 cells could initiate the OCs differentiation cascade through the release of IL-17/RANKL molecular storm, thereby contributing to OP. The migration efficiency of these cells is further enhanced by the intestinal barrier disruption following ovariectomy (OVX).^101,126,149 Of particular interest is the presence of metabolic checkpoints in this pathological migration process. Tryptophan metabolites, such as IAA and IPA, act through the AhR signaling pathway to facilitate the transition from the M1 phenotype to the M2 phenotype. This shift induced an anti-inflammatory microenvironment predominantly characterized by IL-10, which subsequently establishes a metabolic barrier aimed at limiting the migration of Th17 cells to the bone.⁷⁴ Nevertheless, studies on the gut-brain axis demonstrate a more intricate bidirectional migration pattern. Technological advances such as in vivo phototransformation tracking have greatly facilitated the deciphering of these complex bidirectional migration mechanisms. Chang et al. identified substantial disruption of the gut microbiota following cardiac arrest, characterized by abnormal proliferation of Enterobacteriaceae and subsequent LPS release. This event triggered the TLR4-triggered receptor expressed on myeloid cells 1 (TREM1) signaling pathway, resulting in a substantial migration of small intestinal macrophages that highly express the TREM1 to the brain parenchyma, where they initiate neuroinflammatory responses.¹⁰² And altered gut microbiota promoted monocyte infiltration into the ischemic brain area by activating the TLR4/CCL2 axis post-stroke. Furthermore, disruption of the SCFAs-GPR43 signaling axis alleviated the migration restraint on TCRαβ⁺ T cells, enabling their traversal across the blood-brain barrier via the CXC chemokine ligand (CXCL) 11/CXCR3 pathway.^103,104,150 Notably, specific targeting of Th17 cell gut homing by blocking the α4β7-integrin and its ligand mucosal vascular addressin cell adhesion molecule 1 pathway impaired T cell migration to the large intestine, indicating a significant evolutionary connection between neuroimmune and mucosal immune systems.¹⁵¹ Besides, Clostridium butyricum facilitated targeted migration of Treg cells to pancreatic lymph nodes by inducing PPARγ-Foxp3 epigenetic modifications during immune interventions in metabolic diseases. This process was driven by alterations in chromatin accessibility and mediated through the inhibition of HDACs.¹⁰⁵ Furthermore, washing microbiota transplantation (WMT) has been shown to upregulate the expression of CXCR6 on innate lymphoid cells (ILCs) 3 in the mouse liver, thereby promoting their migration to the liver via the CXCL16/CXCR6 signaling axis.¹⁰⁶ The aforementioned sophisticated regulatory mechanisms ensure the precise migration of immune cells from the gut to disease sites and their subsequent functional execution.

Innate immune response

ILCs

ILCs, as crucial components of the innate immune system, possess a functional repertoire remarkably similar to that of Th cells despite the absence of specific antigen receptors characteristic of adaptive immunity. These diverse cells are predominantly located within mucosal barrier tissues, with a particularly prominent role as “immune sentinels” in the intestinal microenvironment. They are essential for maintaining intestinal immune homeostasis by rapidly responding to pathogenic threats and coordinating tissue repair processes. Emerging studies have shown that the functional versatility of ILCs is modulated not only by the immediate microenvironment but also by intricate interactions with the gut microbiota and its metabolic byproducts. This insight provides a novel perspective for studying the role of the gut-immune axis in the pathophysiology of bone-related diseases.^152,153

Based on their secretory profiles and transcriptional regulatory networks, ILCs are categorized into three primary subsets: ILC1s, ILC2s, ILC3s, and ILCregs.¹⁵⁴ Numerous studies have demonstrated that ILCs are closely associated with the pathogenesis of inflammatory diseases such as asthma and colitis. Notably, colitis patients frequently exhibit an increased risk of fracture, which consequently predisposes them to comorbidities such as OP.¹⁵⁵ This might be attributed to ILCs directly driving OCs differentiation by secreting RANKL.^156,157 It is important to highlight that the function of ILCs is intricately regulated by the gut microbiota and its metabolites, and this regulation shows a multi-dimensional characteristic. It has been reported that SFB in the intestines could serve as antigens that are recognized by ILC3s, which subsequently presented to T cells via major histocompatibility complex class II (MHCII), resulting in the activation of ILC3s.¹⁵⁸ Furthermore, intestinal dysbiosis characterized by Candida albicans imbalance has been observed to lead to an increase in the number of ILC2 cells within the pulmonary compartment, which exacerbates allergic airway responses.¹⁵⁹ In addition, metabolites of the gut microbiota could exert immunoregulatory effects on ILCs. Specifically, butyrate significantly ameliorated pulmonary inflammation and airway hyperreactivity by suppressing GATA3 expression in ILC2s.¹⁰⁷ Notably, pravastatin induced IL-33 secretion by intestinal epithelial cells, followed by activation of the IL-33 R on ILC2s, resulting in increased IL-13 production. This cytokine, IL-13, further stimulated self-renewal of intestinal stem cells by activating the Wnt and Notch signaling pathways, thereby contributing to the maintenance of intestinal barrier homeostasis.¹¹⁰ Moreover, in the early stages of life, gut microbiota colonization could inhibit the expression of ten-eleven translocation1 through the bile acid-Takeda G protein-coupled receptor 5 (TGR5) axis, thereby promoting the differentiation of ILC1s and enhancing the ability to resist pathogens.¹¹¹ This phenomenon illustrates the significant impact of the gut microbiota on the epigenetic reprogramming of ILCs. It was found that both commensal bacteria and pathogenic bacteria could stimulate ILC3s to produce IL-22.¹⁶⁰ However, ILC3s, as pivotal regulators of intestinal homeostasis, secrete IL-22, which exhibits a dual functional role dependent on tissue-specific niches, local microenvironmental conditions, and dynamic interactions with complementary cytokine networks.¹⁶¹ For instance, Akkermansia muciniphila promoted IL-22 production by producing SCFAs, thereby contributing to ameliorate colitis.^108,120 Conversely, tryptophan metabolites such as kynurenine and IPA reduced the intestinal inflammatory damage by suppressing IL-22 expression.¹⁶² Furthermore, the secondary bile acid 12-ketolithocholic acid could inhibit IL-17A secretion by activating VDR in ILC3s. This regulatory mechanism not only reduces inflammation but also confers anti-cancer properties through the HDAC inhibition/Sox13 acetylation pathway.^163,164

Huang et al. demonstrated via scRNA-seq that the diminished production of IL-22 by ILC3s is correlated with gut microbiota dysregulation induced by total parenteral nutrition (TPN). By performing 16S rRNA gene sequencing on the gut microbiota of patients with chronic intestinal insufficiency, they observed a significant decrease in the abundance of L. murinus. Particularly importantly, the microbial metabolite indole-3-carboxylic acid, derived from L. murinus, augmented IL-22 secretion by ILC3 by directly interacting and activating RORγt transcription, thereby reinforcing intestinal barrier function and mitigating TPN-induced infectious complications.¹⁶⁵ Beyond the gut microbiota-mediated regulation of ILCs diversity, the dynamic translation among ILCs subpopulations introduces an additional layer of complexity to this multifaceted regulatory network. It has been shown that Helicobacter pylori could induce the transformation of ILC3s into ILC1s, along with enhancing IL-22 and IL-17A production.¹⁶⁶ This indicates that the gut microbiota could modulate the immune balance of the microenvironment by remodeling the phenotypic characteristics of ILCs.

Dendritic cells (DCs)

DCs serve as the “messengers” within the immune system, coordinating the regulation of both innate and adaptive immune responses.¹⁶⁷ Their role is essential in maintaining immune homeostasis and mediating pathological responses. Based on their ontogenetic lineages and functional characteristics, DCs are categorized into two main subsets: conventional DCs (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs.¹⁶⁸ These subsets act as critical bridge between innate and adaptive immunity due to their distinctive antigen-presenting capabilities. In recent years, the dynamic balance mechanism of DCs within the bone microenvironment has attracted extensive attention. The bone microenvironment provides a protective niche for DCs yet inflammatory cues could prompt the transformation of immature DCs into OCs, thereby inducing bone resorption by disrupting the RANKL/OPG axis balance.¹⁶⁹ This provides novel immunological insights into the pathological mechanisms underlying OP, RA, and OA.

Emerging evidence suggests that the gut microbiota and its metabolites are key mediators of the immunomodulatory effects of DCs. SCFAs exert dual regulatory effects on DCs function by inducing epigenetic reprogramming and cytoskeletal remodeling. They were capable of downregulating the expression of inflammation-related genes, such as IL-6 and IL-12, as well as reducing the secretion of chemokines like CCL5 and CXCL9, thus modulating DCs activity and recruitment.¹¹³ Furthermore, SCFAs inhibited HDACs and subsequently activated the src family kinase/PI3K/Rho family GTPases signaling pathway, facilitating actin polymerization-driven dendrite extension and significantly enhancing antigen capture efficiency.¹¹² In contrast, pyruvate selectively stimulated the intestinal cDC1 subset via the GPR31 receptor, resulting in the formation of trans-epithelial dendrites that directly intercept antigens in the intestinal lumen. This process is strongly associated with the activation of CD8⁺ T cells.¹⁷⁰ Furthermore, bile acid metabolites derived from the gut microbiota are likewise involved in the immunoregulatory effects on DCs. For instance, both DCA and LCA could inhibit the activation of NF-κB within DCs by regulating the TGR5-cyclic adenosine monophosphate-protein kinase A signaling pathway, which in turn reduce inflammatory responses.¹¹⁵ Meanwhile, LCA interacted with the TGR5 receptor to inhibit glutathione synthesis and induced oxidative stress, which could compromise the antigen-presenting capacity of DCs and suppress Th1/Th17 responses.¹¹⁶ In contrast, DCs deficient in TLR5 promoted the proliferation of CD4⁺ T cells and the secretion of cytokines, including pro-inflammatory mediators such as TNFα and IL-6, thereby exacerbating the inflammatory response.¹¹⁷ Additionally, 3β-Hydroxydeoxycholic acid modulated the metabolic phenotype of DCs by antagonizing the FXR, thus promoting the generation of Treg cells.¹¹⁴ Interestingly, it is common for gut microbiota to affect the function of other immune cells by regulating DCs. It has been demonstrated that the polysaccharide A from Bacteroides fragilis, upon binding to TLR2, activated pDCs, which subsequently engaged with naïve CD4⁺ T cells via MHCII, CD86, and inducible co-stimulator-related factor on their surface. This resulted in the expansion of IL10⁺ Treg cells and the suppression of intestinal inflammation.¹¹⁹ In contrast, L-lysine, produced by the metabolism of Dubosiella newyorkensis and Clostridium innocuum, enhanced the immunotolerant phenotype of DCs via the AhR-IDO1-kynurenine axis. This mechanism provided dual protective benefits in the dextran sodium sulfate-induced mouse colitis model by inhibiting Th17 cells activation and enhancing intestinal barrier function.¹¹⁸ And the dihydrolipoamide acetyltransferase (DlaT) protein located within the S-layer of Propionibacterium strain is instrumental in promoting Th17 cell differentiation. By binding to the SIGlec-containing receptor-1 on DCs, the DlaT protein initiated Th17 cell differentiation signaling pathways, thus contributing to the amplification of inflammatory responses.¹⁷¹ It is noteworthy that the type I interferon (IFN-I) signaling network serves as a pivotal “immune initiation switch” within the gut microbiota-DCs-T cell axis. Upon stimulation by the gut microbiota, pDCs persistently secreted low levels of IFN-I, which were essential for maintaining cDCs in a “pre-activated alert” state through interferon receptor. Meanwhile, this baseline activation was indispensable for cDCs to respond to pathogenic stimuli and initiated T cell immune responses. In scenarios where IFN-I signaling was inhibited or pDCs were depleted, cDCs failed to effectively activate T cell responses despite the presence of direct stimulatory signals. This highlights the hierarchical dependence of signal transduction associated with microbiota regulation of dendritic cell function.¹⁷²

Adaptive immune response

B cells

The specific regulation of the immune system is fundamentally based on the principle of “self-non-self” recognition, and B cells are the central agents of humoral immunity.¹⁷³ In the bone microenvironment, B cells play a dual role in maintaining bone homeostasis. They not only stimulate OC formation through the excessive expression of TNF, RANKL, and CCL3 but also could inhibit OC differentiation via the ERK/NF-κB signaling pathway.¹⁷⁴ The potential interaction mechanism underlying the transition of this functional phenotype in relation to the gut microbiota is a particularly intriguing aspect of this research.

Furthermore, there is an increasingly well-documented co-evolutionary relationship with the gut microbiota, as demonstrated by the development and functional adaptation of B cells. The relationship between them was elucidated through scRNA-seq. The absence of gut microbiota may result in abnormalities in altered quantities and functional defects of B cells.¹²⁵ The microbiota has been demonstrated to regulate a wide-ranging network that is integral throughout all stages of the B-cell lifecycle, from bone marrow precursor stages to terminal plasma cell differentiation. The microbiota orchestrates these regulatory processes via metabolic reprogramming and epigenetic modification.^175,176 Recent research has revealed that the development of B cells is subject to epigenetic programming influenced by gut microbiota metabolites, with these influences being evident from the fetal stage onwards.¹⁷⁷ Notably, neonatal regulatory B (Breg) cells, a distinct B cell population specific to fetal and neonatal development, exert negative regulation on IgM production via autocrine IL-10 secretion. scRNA-seq of neonatal Breg cells demonstrated that the antibodies they generate display extensive bacterial reactivity, capable of recognizing microorganisms from six different bacterial phyla present in the early gut microbiota, including Firmicutes and Bacteroidetes.¹⁷⁸ It demonstrated a significant interaction between the gut microbiota and the functioning of B cells. Emerging evidence has shown that the gut microbiota could stimulate the development of IL-10-producing Breg cells via the TLR2/MyD88/PI3K signaling pathway, thereby contributing to colonic stability and preventing the progression of inflammatory bowel disease.¹⁷⁹ Nevertheless, in instances of intestinal dysbiosis, an abnormal distribution of B cells was observed, which correlates with increased levels of LPS. Specifically, LPS activated the TLR4 signaling pathway, increasing cellular sensitivity to the Fas ligand and inducing apoptosis in conventional memory B cells. Simultaneously, LPS upregulated the expression of co-stimulatory molecules, including MHCII and CD86, facilitating the activation and the proliferation of naive B cells. This cascade of events ultimately resulted in the disruption of mucosal immune homeostasis, diminishing the ability to defend against pathogens and exacerbating the development of autoimmune diseases. However, this adverse scenario could be alleviated through the intervention of SCFAs.¹⁸⁰ SCFAs not only facilitated the differentiation of Breg cells through FFAR2-mediated STAT3 phosphorylation, thereby alleviating RA.¹²¹ Furthermore, SCFAs exhibited a dual regulatory influence on B cell function via the 5-HIAA-AhR axis. Specifically, SCFAs could activate AhR-dependent transcriptional pathways to enhance the immunosuppressive capabilities of Breg cells, while simultaneously inhibiting Blimp-1 expression to prevent the terminal differentiation of germinal center B cells into plasma cells.¹²² This mechanism of metabolic regulation provides novel molecular insights into the gut microbiota-mediated regulation of B cell fate determination.

T follicular helper (Tfh) cells

Tfh cells contribute to humoral immunity, promoting B cell activation, proliferation, somatic hypermutation, and class-switching recombination, ultimately leading to the production of plasma cells and memory B cells capable of producing high-affinity antibodies.^181,182 The germinal center serves as a critical site for B cell differentiation and antibody production, wherein Tfh cells are essential for both the establishment and the functional efficacy of this center. Emerging studies have shown that the activation and proliferation of B cells within the lymph nodes of RA patients are intrinsically dependent on the support of Tfh cells, regardless of the disease activity status. Furthermore, Tfh cells facilitated their migration toward B cell follicles via a CXCR5-mediated chemotactic pathway. However, excess of Tfh cells could precipitate heightened germinal center activity, promoting the development of autoreactive B cells and increasing the production of autoantibodies, thereby significantly promoting the onset and progression of RA.^183,184

The supportive effect of Tfh on B cells is inseparable from the regulation of gut microbiota.¹⁸⁵ The gut microbiota-derived bile acid metabolite glycochenodeoxycholic acid (GLCA) has been identified as a crucial inducer of Tfh cell activation via the CXCL13 signaling pathway.¹⁸⁶ Moreover, recent studies have highlighted the profound impact of specific gut microbiota components, such as SFB, on the immune system. SFB modulated the expression of the pivotal transcription factor Bcl-6 in CD4⁺ T cells within the intestinal Peyer’s patches by suppressing the IL-2 signaling pathway. This regulation facilitated the differentiation and migration of Tfh cells, thereby contributing to the elevated production of autoantibodies and the exacerbation of RA. Notably, SFB-induced Tfh cell responses were initiated prior to the onset of arthritis and were indispensable for the progression of RA mediated by SFB. Furthermore, DCs have been implicated in the SFB-mediated suppression of IL-2 Rα, highlighting the intricate interactions between the gut microbiota and the immune system.¹²³

In conclusion, gut microbiota play a crucial role in the polarization, chemotaxis, and differentiation of immune cells. Firstly, the gut microbiota could sustain the equilibrium of immune cell polarization via its metabolites, thus regulating inflammatory responses. Furthermore, the interaction between gut microbiota and systemic organs is contingent upon the modulation of immune cell migration. Additionally, the gut microbiota exerts an impact on both innate and adaptive immune responses. This suggested a close and harmonious “cooperative journey” between the gut microbiota and the human immune system, collectively establishing a robust fortress to protect human health. However, despite the growing evidence highlighting the functional significance of the gut microbiota in immune system regulation, the intricate mechanisms remain poorly understood, and clinical applications necessitate further integration with disease contexts. Consequently, future studies utilizing high-precision techniques such as metabolomics, scRNA-seq, and 16S rRNA gene sequencing are anticipated to uncover the underlying mechanisms of interaction between the gut microbiota and the immune system under both physiological and pathological conditions, thus facilitating the development of “personalized microbiota-targeted” therapeutic approaches for immune-related diseases.

Regulatory mechanism of gut microbiota on bone microenvironment

In recent years, the gut microbiota has emerged as a novel paradigm to investigate the underlying mechanisms of bone homeostasis imbalance through the establishment of a bidirectional gut-bone axis as a biological communication channel.¹¹ This axis transcends traditional organ boundaries by establishing integrative connections across organs, thus forming multidimensional interactive networks. Mechanistically, the gut microbiota dynamically coordinates OCs-OBs coupling balance through multilevel pathways including metabolite-driven regulation, the secretion of vesicles, and intervention of other “organ-bone” axis to co-regulate the bone microenvironment Figure 3. Furthermore, synergistic interactions between gut microbiota and stromal cells, such as fibroblasts and chondrocytes, significantly contribute to bone homeostasis maintenance.¹⁸⁷ Notably, the diverse functional properties of gut microbial metabolites provide promising therapeutic targets for precise interventions in bone metabolic diseases.

Figure 3.

Gut microbiota-mediated modulation of the bone microenvironment.

The bone microenvironment, comprising OBs, OCs, chondrocytes, synovial fibroblasts, and vascular endothelial cells, serves as the fundamental basis for maintaining bone metabolic equilibrium and skeletal integrity. The gut microbiota exerts regulatory control over cellular activities within this specialized niche through the secretion of metabolites and BEVs, which subsequently impact bone homeostasis. Furthermore, the gut microbiota indirectly influences bone remodeling via multiorgan crosstalk networks, including the vascular-bone, liver-bone, and brain-bone axes, demonstrating systemic coordination in bone regulation. Created with BioRender.com

Gut microbiota-derived metabolite-driven the regulation of the bone microenvironment

OBs, the central effector cells in bone remodeling, are responsible for the construction of the mineralized bone matrix through the secretion of matrix components, including type I collagen and osteocalcin. In contrast, OCs are responsible for the degradation of the bone matrix through the secretion of acids and enzymes, such as cathepsin K and tartrate-resistant acid phosphatase (TRAP).¹⁸⁸ Additionally, chondrocytes, fibroblasts, and vascular endothelial cells are also key contributors to the bone microenvironment.

It has been demonstrated that the gut microbiota bidirectionally modulates the gut-bone axis through its metabolic products, significantly affecting the OCs-OBs coupling equilibrium. SCFAs have been demonstrated to exert a favorable influence on OP by impeding the differentiation of OCs. This was accomplished through the attenuation of the IFN signaling pathway and the modulation of inflammatory signals, including the negative regulatory factor dual-specificity phosphatase 1 in OCs.¹⁸⁹ Specifically, valproic acid reduced the expression of pro-inflammatory cytokines through the inhibition of RELA protein while simultaneously upregulating anti-inflammatory mediators such as IL-10. This dual regulatory mechanism effectively suppressed OCs differentiation and enhanced the mineralization potential of OBs precursor cells.¹⁹⁰ Furthermore, butyric acid mitigated joint inflammation and bone destruction in CIA mice by selectively inhibiting HDAC2 in OCs and upregulating the bone-protective protein secretory leukocyte protease inhibitor.⁸⁶ Besides, Clostridium sporogenes generated IPA, which activated pregnane X receptor to prevent nuclear factor of activated T cells c1 nuclear translocation, thereby inhibiting RANKL-induced OCs differentiation.¹⁹¹ Moreover, indole sulfate inhibited OB differentiation and bone formation by inhibiting the expression of the key OB marker Runx2 through the AhR/p38MAPK signaling axis during gut microbiota dysbiosis.¹⁹² This bone homeostasis imbalance was exacerbated by LPS from Gram-negative bacteria. When LPS penetrated the intestinal barrier, it instigated the activation of the NLRP3 inflammasome within OBs via the TLR4/MyD88 signaling pathway. It is hypothesized that this activation has the potential to induce caspase-1-dependent pyroptosis and impaired bone formation, which may contribute to OP.¹⁹³ Additionally, during pregnancy, maternal LPS affected distal-less homeobox 5 expression via the CYP1B1-retinoic acid pathway in the fetal liver, triggering transgenerational OBs dysfunction.¹⁹⁴

The secretion of extracellular matrix mediated by chondrocytes is essential for the construction and maintenance of cartilage, and this process is also regulated by the gut microbiota. Butyric acid has demonstrated therapeutic potential for OA by inhibiting the PI3K/AKT/mammalian target of rapamycin signaling pathway. This inhibition enhanced autophagy activation in chondrocytes, resulting in increased autophagic flux. Consequently, the degradation of the extracellular matrix was reduced, and apoptosis in chondrocytes exposed to IL-1β was decreased, contributing to the treatment of OA.¹⁹⁵ In addition, urolithin B, another metabolite derived from the gut microbiota, was found to possess multifaceted properties that contributed to the alleviation of OA. It not only inhibited NF-κB-mediated inflammatory responses but also reduced the expression of degradation enzymes such as matrix metalloproteinase 3. Furthermore, urolithin B has been shown to promote the synthesis of type II collagen and proteoglycans, thereby inhibiting the degradation of cartilage, and ultimately providing relief from OA.¹⁹⁶

Fibroblast-like synoviocytes (FLS), as the primary cellular component of the synovium, are integral to the maintenance of joint function by synthesizing extracellular matrix components and lubricants.¹⁹⁷ Conversely, in inflammatory diseases such as RA, FLS undergo pathological proliferation and phenotypic alterations.¹⁹⁸ Through both direct and indirect destructive mechanisms, they emerged as central mediators of chronic inflammation, exacerbating inflammatory cascades and driving articular cartilage degradation. Emerging evidence suggested that the regulatory function of FLS in maintaining the bone microenvironment was also regulated by gut microbiota-driven signaling pathways, further highlighting the gut microbiota in inflammatory joint diseases. It has been reported that propionic acid produced by Bacteroides fragilis serves multiple beneficial roles in RA and OA. In particular, it functioned as an inhibitor of HDAC3, thereby increasing the acetylation level of forkhead box k1, which resulted in the inactivation of RA-FLSs and subsequently ameliorated RA.¹⁹⁹ Besides, propionic acid could inhibit the migration and senescence of synovial fibroblasts, thereby alleviating OA.²⁰⁰ It is noteworthy that MH7A cells isolated from the RA-FLS of RA patients could promote synovial hyperplasia and cartilage destruction by releasing pro-inflammatory factors. In vitro experiments have demonstrated that Saccharomyces boulardii could inhibit the migration of MH7A cells and promote their apoptosis, thereby suppressing the onset and progression of arthritis.²⁰¹

Bone circulation is indispensable for mediating intercellular communication within the bone microenvironment and its crosstalk with external systems. As critical components of bone circulation, vascular endothelial cells are essential for regulating hematopoiesis, osteogenesis, and osteoclastogenesis.²⁰² Furthermore, gut microbiota-derived metabolites regulate vascular endothelial cell functionality, a regulatory mechanism critical for maintaining homeostasis within the bone microenvironment. It has been reported that D-malate, produced by the gut microbiota, possessed the capability to diminish the proliferation of vascular endothelial cells and suppress angiogenic processes. The underlying mechanism of this effect was contingent upon the elevation of intracellular acetyl-CoA levels and the acetylation of Cyclin A.²⁰³ Nevertheless, TMAO has been shown to stimulate vascular endothelial cells, promoting arterial thrombosis.²⁰⁴ TMAO also exacerbated mitochondrial dysfunction and reactive oxygen species (ROS) production by upregulating the expression of succinate dehydrogenase complex subunit B within these cells, thereby inducing pyroptosis.²⁰⁵ In addition, the endothelial-to-mesenchymal transition observed in vascular endothelial cells was mediated by TMAO through the activation of the endoplasmic reticulum stress kinase PERK.²⁰⁶ The combined effects of these pathways on vascular endothelial cells may result in insufficient perfusion and metabolic disturbances within bone tissue. Interestingly, tryptophan metabolites derived from the gut microbiota exert a dualistic influence on the vasculature of RA patients. It has been demonstrated that IAld promoted endothelial tube formation, whereas IAA inhibited this process.²⁰⁷ This finding challenges the conventional understanding of the pathogenic mechanisms underlying the gut-bone axis.

Regulation of extracellular vesicles derived from gut microbiota

Bacterial extracellular vesicles (BEVs) serve as innovative nanoscale communicators within the dynamic interactions between microorganisms and their hosts, highlighting their distinctive biological significance in the regulation of bone homeostasis. These nanoscale membrane structures, with diameters ranging from 20 to 400 nm, adeptly encapsulate bioactive molecules such as proteins and nucleic acids.²⁰⁸ Beyond their role in facilitating intermicrobial communication, BEVs also functioned as crucial molecular mediators through which the gut microbiota orchestrates the regulation of distant bone homeostasis.²⁰⁹ The structural alterations in the bacterial cell wall impart distinct biological and genetic characteristics to BEVs. Outer membrane vesicles (OMVs) from Gram-negative bacteria, with a diameter ranging from 20 to 250 nm, originate from the budding of the outer membrane and are enriched with periplasmic proteins and cytoplasmic components. In contrast, cytoplasmic membrane vesicles from Gram-positive bacteria, formed by the cytoplasmic membrane invagination, could reach diameters up to 400 nm and directly encapsulate cytoplasmic material.²¹⁰

The intrinsic structural differentiation endows them with specialized molecular delivery capabilities, positioning them as natural vehicles for precise therapeutic interventions in bone tissue. For instance, extracellular vesicles (EVs) from L. animalis mitigated GC-induced deficits in osteogenic differentiation through a dual mechanism. Firstly, they directly infiltrated OBs, promoting their differentiation and stimulating the secretion of osteogenesis-associated factors, such as BMP, thereby enhancing osteogenic activity. Secondly, they induced endothelial cells to release pro-angiogenic factors, such as vascular endothelial growth factor, which facilitated the restoration of the local bone microvascular network through paracrine signaling pathways.²¹¹ Notably, EVs derived from various gut microbiota exhibit distinct specificities. Lactobacillus rhamnosus GG (LGG) EVs were recognized for promoting vascular calcification and thrombosis through the activation of the PI3K/AKT pathway.²¹² These circulatory impairments have the potential to exacerbate ischemic damage within bone tissue. During growth and development, it has been observed that EVs originating from the maternal gut microbiota were capable of penetrating the placental barrier and reaching the fetus.²¹³ This finding suggests a potential mechanism through which early maternal factors could influence fetal skeletal development via EVs. However, many studies on the intestines of children have shown that EVs released from Akkermansia muciniphila could infiltrate and accumulate in bone tissue. These EVs contributed to anti-osteoporotic effects by stimulating OBs generation and concurrently suppressing OCs formation.²¹⁴ While Liu et al. did not thoroughly explore the specific mechanisms of action of Akkermansia muciniphila-derived EVs in the treatment of OP, it is expected that future research will address this issue through gene therapy and pharmacological interventions. Notably, it has been demonstrated that the potential mechanisms by which Proteus OMVs ameliorate bone-related diseases such as OP and RA. Specifically, Proteus OMVs have been shown to exert a protective influence on bone by modulating the expression of specific molecular targets in OCs. They accomplished this by downregulating miR-96-5p and upregulating ATP-binding cassette subfamily A member 1, which collectively inhibit osteoclastogenesis and promote apoptosis in these cells, thereby enhancing bone formation.²¹⁵ Surprisingly, BEVs function as efficient nanocarriers capable of delivering bacterial products to distant tissue cells, thereby enabling microbe-host communication. On the one hand, pathogen-associated molecular patterns contained within the BEVs cargo can bind to pattern recognition receptors on host cells, activating downstream inflammatory signaling pathways and contributing to synovitis and cartilage degradation. On the other hand, probiotic-derived BEVs can mitigate inflammatory responses by releasing anti-inflammatory metabolites and probiotic surface proteins, thus promoting intestinal and joint health. Therefore, BEVs hold potential as drug delivery systems that regulate intestinal permeability and modulate immune-inflammatory responses via the gut-joint axis, ultimately enhancing the therapeutic efficacy for OA.²¹⁶

Intervention of “organ-bone” axis

The concept of the gut-bone axis has been greatly expanded beyond its initial description as a bidirectional regulatory network characterized by direct interactions between the gut microbiota and bone. The gut-bone axis is now considered a multidimensional system that integrates the synergistic interactions of various axes, including the vascular-bone, liver-bone, and brain-bone axes.^217–219 Thus, dissecting the regulation of other organ-bone axes by gut microbiota is of great significance for bone homeostasis maintenance.

The circulatory system, as a fundamental platform for interorgan communication, is a critical channel for the gut microbiota to remotely regulate bone homeostasis.²²⁰ Vascular endothelial cells maintain the stability of the bone microenvironment by dynamically regulating vascular permeability, hemodynamic characteristics, and intercellular signaling pathways.²²¹ However, gut microbiota-induced vascular calcification may disrupt the balance of bone homeostasis. It has been demonstrated that TMAO induced the expression of the inflammatory cytokine IL-1β by activating the NLRP3 inflammasome and the NF-κB signaling pathway, which in turn promoted the osteogenic differentiation of vascular smooth muscle cells, resulting in vascular calcification.²²² Furthermore, gut microbiota-derived metabolites transported through the portal venous circulation activate hepatic innate immunity, thereby establishing a regulatory pathway for bone homeostasis. LPS from the gastrointestinal tract activated hepatic DCs via the TLR2 signaling pathway, resulting in pro-inflammatory cytokine secretion, especially TNF-α and IL-6. These cytokines circulated to the bone microenvironment, where they promote the differentiation of Th17 cells via the RANKL/RANK/OPG signaling cascade, ultimately enhancing OC activity.²²³ It is worth noting that the liver, as an endocrine organ, synthesizes insulin-like growth factor 1 (IGF-1), and its production was also modulated by the gut microbiota.²²⁴ IGF-1 levels in serum of GF mice decreased by 43.7% compared to normal mice, whereas probiotic supplementation restored these levels to 82.3% of normal values.²²⁵ Furthermore, another study demonstrated that IGF-1 contributed to maintaining the dynamic balance of bone homeostasis by stabilizing β-catenin and enhancing the activity of Wnt-dependent signaling pathway, thus reducing OBs apoptosis and promoting osteogenesis.²²⁶

Parathyroid hormone (PTH) is a key regulator of the neuroendocrine-bone axis and is implicated in the regulation of the skeletal system, a process that is closely linked to the gut microbiota. Specifically, PTH binding to the PTH-PTHrP receptor leads to the expansion of TNF⁺ T cells and Th17 cells within the gut, followed by migration to the bone microenvironment for promoting bone resorption and inhibiting bone formation. However, PTH-induced bone loss was observed only in the presence of SFB-rich gut microbiota, a phenomenon associated with the ability of SFB to induce Th17 cell proliferation.¹⁰¹ In addition, the brain, as the most crucial organ in the human body, also relies on the intervention of the gut microbiota to regulate the skeletal system. Among the neurotransmitters, serotonin is considered to be a pivotal factor in regulating bone development and maintaining bone metabolism stability. It has been found that Clostridium butyricum could significantly increase serotonin and dopamine levels, as well as upregulate signaling pathways related to bone metabolism, including the signaling pathways of glucagon, insulin, and PI3K/AKT, thus promoting bone formation and preventing bone resorption.²²⁷ In contrast, a decrease in 5-HT levels resulted in a significant increase in bone mass, which was associated with a decreased presence of OCs on the bone surface.²²⁸ Further research has demonstrated that reducing 5-HT levels could prevent bone loss in OVX mice.²²⁹ Consequently, this evidence suggests that the brain indirectly affects the gut-bone axis, playing a crucial role in regulating bone homeostasis through the gut microbiota.

In summary, the gut-bone axis offers a novel perspective for investigating the mechanisms and developing therapies for bone-related diseases through the interaction between the gut microbiota and the bone microenvironment, encompassing microbial metabolites, microbiota-derived exosomes, and the organ-bone axis. Although microbiota-derived exosomes demonstrate significant therapeutic potential in modulating bone metabolism, their clinical translation in bone-related diseases faces challenges due to insufficient stability and current limitations in isolation and purification techniques. Therefore, to further explore the mechanisms of the gut-bone axis and develop microbiota-targeted therapies for bone-related diseases, future research should prioritize the molecular mechanisms underlying the gut microbiota’s regulation of bone homeostasis, and conduct broader clinical trials to assess the safety and efficacy of these interventions.

Bridging role of immune cells in the gut-bone axis

Recent advances in the gut-immune axis have identified gut-coordinated immunomodulation as a central mechanism in the pathogenesis of bone-related diseases (Figure 4). While preliminary evidence confirms gut-immune-bone crosstalk, critical questions remain regarding its autonomy as an independent regulatory network and underlying signaling pathways. Emerging research describes this axis as a tripartite continuum that combines gut microbiota-driven immunomodulation with bone homeostasis, providing a framework for redefining therapeutic strategies for bone-related diseases through microbiome-immune-bone synergy.²³⁰

Figure 4.

Gut-immune-bone axis regulatory mechanisms.

Under physiological conditions (left panel), gut microbiota-derived metabolites traverse the intestinal barrier and enter systemic circulation or local tissues, modulating immune cell activity. These immunomodulatory interactions preserve immune homeostasis, thereby sustaining balanced bone remodeling through coordinated OCs-OBs coupling. Conversely, in pathological states (right panel), dysbiotic microbiota exhibit depletion of commensal probiotics and expansion of pathobionts, resulting in compromised tight junction integrity and increased intestinal permeability. This barrier dysfunction enables pathogenic species to release cytotoxic metabolites that trigger systemic immune dysregulation. Such perturbations amplify osteoclastogenesis, suppress osteogenesis, and accelerate cartilage degradation by chondrocytes, ultimately driving the pathogenesis and progression of bone-related diseases including RA, OA, and OP. Created with BioRender.com

Treg/Th17

Bone-related diseases primarily originate from pathological imbalance between bone formation and bone resorption processes, which are intricately linked with immune system dysregulation. It has been elucidated that the balance between Treg/Th17 is crucial for maintaining bone homeostasis, primarily mediated through their regulation of OC differentiation and inflammatory responses. However, the host immune system exhibits dual regulatory capacity in bone remodeling processes. A shift toward Th17 cells-dominant in the Treg/Th17 ratio, characterized by Th17 cell expansion and Treg cell depletion, contributes mechanistically to the degradation of articular cartilage and bone destruction. Specifically, Th17 cells exacerbated inflammatory responses through the secretion of IL-1/IL-22 while promoting OC differentiation, thereby enhancing bone resorption. Conversely, Treg cells suppressed OC activity and inflammation via IL-10/TGF-β production, and simultaneously supported osteogenic differentiation through paracrine signaling.²³¹ Therefore, Treg/Th17 balance is an important pathway for the regulation of bone homeostasis by the gut microbiota.

The gut microbial ecosystem exerts hierarchical control over Treg/Th17 balance, thereby regulating bone remodeling dynamics.²³² LGG was capable of improving the Treg/Th17 balance in mesenteric lymph nodes and bone marrow, reducing the proportion of Th17 cells and increasing that of Treg cells, promoting bone formation to ameliorate OP. This might involve the mediation of metabolites from the gut microbiota.²³³ For instance, SCFAs exhibit dual osteoprotective effects by regulating Treg/Th17 balance. They could both enhance the accessibility of Foxp3 chromatin in Treg cells by inhibiting HDAC, and promote Treg cell expansion as well as function based on FFAR2 agonism to inhibit OCs differentiation.²³⁴ Additionally, the regulatory role of secondary bile acids in bone metabolism through the Treg/Th17 balance has also been established. Substantial studies have revealed that PMO patients exhibited decreased gut microbiota diversity, and there was a negative correlation between their fecal LCA levels and bone density. Mechanistically, the derivative of lithocholic acid as well as 3-oxoLCA, has been demonstrated to directly bind to RORγt, thereby inhibiting the differentiation of Th17 cells. Meanwhile, isoalloLCA facilitated the differentiation of Treg cells by inducing Foxp3 expression via mitochondrial ROS, which subsequently aided in inhibiting inflammatory processes.^92,93 Furthermore, GLCA enhanced the number of Treg cells through the activation of the constitutive androstane receptor (CAR), thereby facilitating the osteogenic differentiation of mesenchymal stem cells and contributing to the alleviation of PMO.⁹² Tryptophan metabolites derived from the gut microbiota have also been shown to modulate the Treg/Th17 balance through immunoregulatory mechanisms. The bacterium L. murinus could elevate the levels of IA and IAld in the serum. The improvement in symptoms of RA patients was associated with tryptophan metabolites, including IA and IPA, which activate the AhR, leading to an increase in Treg cells and a decrease in Th17 cells, thus alleviating inflammation.^91,94 Besides, Eggerthella lenta activated Th17 cells by metabolic immunomodulatory factors, such as the Th17 cell differentiation inhibitor RORγt, which was related to the presence of the strain-specific enzyme Cgr2.²³⁵ It has also been found that the long-term use of antibiotics in mice resulted in a decreased gut microbiota diversity and a lowered abundance of bacteria associated with gut health. Nevertheless, the abundance of Proteobacteria increased significantly. Such an imbalance of the gut microbiota might disrupt intestinal homeostasis, allowing bacteria or their metabolites to enter the bloodstream. The subsequent result was an imbalance of Treg/Th17 in the periodontal tissues, causing systemic inflammation, intensifying of periodontal tissue destruction and alveolar bone loss.²³⁶

Immunoregulatory network interactions are further involved in the regulation of Treg/Th17 balance by gut microbiota. For instance, Clostridium pruszechii elicited a tolerant DCs phenotype by activating TLR2/6, driving IL-27/CD39/IDO1-dependent Treg cell differentiation that inhibited osteoclastogenesis.^237,238 In contrast, P. copri dysbiosis in RA could enhance DCs immunogenicity through MHCII/CD86/CD80 upregulation, promoting arthritic Th17 cell responses against bacterial antigens but induce potential joint damage.²³⁹ Functional plasticity of Th17 cells goes beyond pro-inflammatory effects. CCR6⁺ Th17 cells executed gut-to-bone trafficking via CCL20 chemotaxis, locally driving IL-17A/RANKL-mediated OC activation and joint destruction. IL-17/IL-17RA axis activation recruited neutrophils, amplifying the osteoclastogenic cascade and inflammatory bone loss.²⁴⁰ Surprisingly, commensal bacteria-induced regulatory Th17 cells restricted effector T cells via the IL-10/CCAAT/enhancer binding protein MAF pathways, and intestinal macrophage crosstalk mediated immunosuppression of the lamina propria.²⁴¹

Breg

As a crucial component of adaptive immunity, B cells participate in the immune response through antibody production and antigen presentation. Additionally, the heterogeneity and functional plasticity of B cell subsets have attracted much attention in recent years.²⁴² Breg cells have emerged as a distinct immunomodulatory subset of B cells, which maintain immunosuppression by secreting cytokines such as IL-10 and TGF-β. Dysfunction in Breg cells is closely associated with the development of autoimmune diseases.²⁴³ Compared to Treg cells, Breg cells lack lineage-defining transcription factors, such as Foxp3, and their phenotypic dynamic characteristics suggest that they may represent functional states rather than independent lineages, which poses unique challenges to understanding their regulatory mechanisms.²⁴⁴ It has been found that Breg cells could inhibit RANKL-induced osteoclastogenesis by secreting IL-10, thereby ameliorating bone loss.²⁴⁵ Furthermore, another study showed that the gut microbiota was able to promote the differentiation of Breg cells in the spleen and mesenteric lymph nodes, enhance IL-10 release, and subsequently suppress the inflammatory response.²⁴⁶ This provides compelling evidence supporting the efficacy of the gut-Breg-bone axis.

Similar to other mechanisms of regulation of immune cells by gut microbiota, gut microbiota-derived metabolites also play an important role in Breg cell regulation.²⁴⁷ Studies have revealed that the probiotic Bacillus coagulans (BC) could enhance the production of SCFAs in the intestine, with a particular emphasis on butyric acid, thereby increasing the number and functionality of Bregs, promoting the release of IL-10, inhibiting osteoclastogenesis and bone resorption, and thus decelerating bone loss.²⁴⁸ Gut microbiota-derived acetic acid could also promote the secretion of IL-10 by Breg cells and reduce the severity of arthritis. However, the reprogramming effect of acetic acid on Breg cells is different from the mechanism by which acetic acid promotes Treg cell differentiation. It was not dependent on the activation of GPR43, but on promoting the degree of lysine acetylation.²⁴⁹ 5-HIAA could directly activates AhR to enhance the immunosuppressive function of Breg cells, thereby inhibiting the progression of arthritis.¹²² Besides, the regulation of Breg cells by gut microbiota also involves crosstalk with other immune cells. BC can significantly ameliorate PMO, which is associated with its modulation of the “Breg-Treg-Th17” cell axis. In addition to regulating the anti-osteoclastogenic function of Bregs, BC could enhance the immunosuppressive and immunomodulatory functions of Bregs. Consequently, it inhibits the differentiation of Th17 cells and promotes the differentiation of Tregs, thereby alleviating inflammatory responses and bone loss.²⁴⁸ Notably, in antigen-induced arthritis models, gut-associated lymphoid tissues (GALT)-derived cytokines including IL-1β and IL-6 promoted the development of Breg cells. Conversely, Breg cells lacking IL-6 R or IL-1R1 reduced IL-10 production and develop more severe arthritis. It was further found that these necessary inflammatory signals for Breg cells were derived from the gut microbiota inducing the production of IL-1β as well as IL-6 by DCs and macrophages in GALT.²⁴⁶ Moreover, Bacillus longum could either directly facilitate Breg-mediated OCs inhibition or restore Treg/Th17 balance to mitigate bone loss.²⁵⁰ It is worth noting that under pathological conditions, IL-10⁺ Breg cells could undergo pathogenic transformation, shifting from cells expressing IL-10 to those expressing RANKL. Yeo et al. identified B cells, rather than T cells, as the primary source of RANKL in patients with RA.²⁵¹ Specifically, IL-10⁺ Breg cells expanded and expressed higher levels of RANKL in the synovial fluid of RA patients, thereby promoting OCs differentiation and exacerbating bone destruction.²⁵² Clinical studies have corroborated these findings. Hu et al. demonstrated that IL-10⁺ Breg cells in RA patients showed a decreased capacity to express IL-10 and were less effective in inhibiting monocyte-mediated secretion of inflammatory factors compared to healthy individuals. Notably, aberrant expression of RANKL by IL-10⁺ Breg cells was observed in both healthy individuals and RA patients, but it was more serious in the latter. However, effective treatment could potentially restore this abnormality.²⁵³ In addition, there is a population of granzyme B (GrB)⁺ Breg cells capable of secreting GrB. Xu et al. found that GrB+ Breg cells have the capacity to exert immunosuppressive function by secreting GrB. This results in TCR chains being down-regulated and T cell apoptosis being induced, in addition to Th1 and Th17 cells being negatively regulated. However, that number of GrB⁺ Breg cells decreased in patients with RA, leading to exacerbation of RA.²⁵⁴

Macrophages

Contemporary research highlights macrophages as central coordinators of bone homeostasis through multifaceted regulatory circuits. The monocyte-macrophage lineage originates from hematopoietic progenitor cells and differentiates into tissue-specific subpopulations that maintain bone homeostasis through phagocytosis, immunomodulation, and cytokine production.^255,256 The emerging paradigm positions the gut microbiota-macrophage-bone axis as essential for tripartite axis functionality, establishing a complex signaling network.

Gut microbiota-dependent M1/M2 polarization regulation is a critical regulator in osteoimmune dynamics. Lactobacillus acidophilus-derived extracellular polysaccharides promoted M2 polarization through the upregulation of CD163 and CD206, further enhancing bone repair through anti-inflammatory and osteogenic properties.²⁵⁷ Similarly, P. histicola inhibited systemic and local inflammatory responses as well as RANKL expression by downregulating the expression of the M1 macrophage marker (CD68) and upregulating the expression of the M2 macrophage marker (CD163), thereby suppressing osteolysis.²⁵⁸ Furthermore, Lactobacillus inhibited osteoclastogenesis, potentially through a mechanism involving the downregulation of M1 macrophage markers (TNF-α, IL-6, and iNOS) and the upregulation of M2 macrophage markers (IL-4, IL-10, and arginase).²⁵⁹ Notably, gut microbiota metabolites are also involved in this process. For instance, tryptophan metabolites such as IAA and IPA reprogramed macrophages toward the IL-10-producing M2 phenotype, promoting osteogenesis and inhibiting osteoclastogenesis.⁷⁴ Nevertheless, the deficiency of P. distasonis would result in the decreased generation of secondary bile acids, attenuate the inhibition of M1 polarization, and ultimately aggravate the occurrence of RA.²⁶⁰ Instead, F. nucleatum was capable of facilitating the infiltration and activation of M1 macrophages and suppressing the polarization of M2 macrophages via the AKT2 signaling pathway.¹⁴³ Pathological M1 polarization disrupts bone remodeling through a dual mechanism. On the one hand, M1 induced a pro-osteoclastogenic cytokine storm, and on the other hand, it suppressed OBs dysfunction mediated by Notch signaling.^261,262 Additionally, the metabolic product TMAO of the gut microbiota promoted the production of mitochondrial ROS and the nuclear translocation of NF-κB, thereby activating the NLRP3 inflammasome and eventually inducing M1 polarization.^146,263 And M1-derived EVs contributed to the decomposition of chondrocytes and resulted in joint destruction through multimodal miRNA signaling, including miR-1246, miR-350-3p and miR-100-5p.^109,264,265

Pyroptosis is a form of programmed cell death, while the pyroptosis of macrophages is closely associated with OCs activity. Moreover, intestinal dysbiosis could trigger caspase-11-mediated macrophage pyroptosis, linking impaired intestinal barrier to osteoimmune dysregulation.²⁶⁶ The NLRP3 inflammasome plays a crucial role in the pyroptosis of macrophages. Pyroptosis-driven IL-1β release activated inflammasomes to establish pro-osteoclastogenic microenvironments.²⁶⁷ It is reported that P. anaerobius could activate NLRP3 inflammasomes in macrophages, thereby inducing Gasdermin D-mediated pyroptosis and secretion of IL-1β, a process associated with TLR2 and TLR4.²⁶⁸ Nonetheless, in a protective capacity, SCFAs and Urolithin A could antagonize the NLRP3 inflammasome and inhibit the pyroptosis-osteoclastogenic coupling to alleviate OP.^267,269

Furthermore, the phagocytic function of macrophages is the first line of defense for the human immune system. Studies have demonstrated that in OVX mice, TRAP particles produced by OCs were phagocytosed by bone tissue macrophages, leading to the formation of a pro-osteoclastogenic microenvironment.²⁷⁰ This phagocytic function of macrophages could be enhanced by the gut microbiota metabolite rhamnose through the activation of the SLC12A4/Rac1/Cdc42 signaling axis.²⁷¹ These findings suggest that the regulation of macrophage phagocytosis by the gut microbiota plays a crucial role in supporting OC-mediated bone resorption.

In conclusion, immune cells, including T cells, B cells, and macrophages, play a bidirectional regulatory role within the gut-bone axis. The gut-immune-bone axis offers novel insights into the development of bone immunology and microbiota-targeted therapies for bone-related diseases, underscoring the critical role of microbe-host interactions in maintaining bone health. However, existing studies predominantly focus on the isolated mechanisms of the gut-immune axis and the gut-bone axis, with limited attention given to the integrated gut-immune-bone axis. A multitude of factors underlie this research gap. Firstly, the communication among the gut microbiota, the immune system, and the skeleton involves multi-system and multi-layered regulation, which complicates the identification of critical regulatory nodes. The diversity of the gut microbiota and inter-individual variability further impede the establishment of causal relationships within the gut-immune-bone axis. Secondly, most studies rely on GF animal models, such as GF mice, whose physiological conditions differ significantly from those of humans. Additionally, in vitro models, such as intestinal organoid-OB co-cultures, remain at a nascent stage of methodological development. Moreover, the majority of studies remain confined to animal experiments or small-scale observational studies, with a lack of large-scale clinical validation. Therefore, future studies should prioritize coherent and in-depth mechanistic research, employing innovative methodologies such as multi-omics integration, organoid systems, and microfluidic chips, to promote the study of the gut-immune-bone axis and the clinical application of microbiota-targeted therapies.

Gut microbiota-focused therapies for bone-related diseases

Traditional therapeutic strategies for bone-related diseases have long been limited by insufficient targeting precision, systemic side effects, and persistent challenges to reverse imbalances in bone homeostasis.^1,272,273 To address these limitations, we propose and elucidate a novel therapeutic strategy that leverages a multidimensional regulatory network involving the gut-immune-bone axis to ameliorate bone-related diseases. The strategy includes probiotic/prebiotic supplementation, FMT, dietary modifications, and pharmacological interventions (Figure 5). Focusing on the dysregulation of the gut microbiota as a pivotal regulatory node, this approach aims to rectify immune system imbalances and further target the fundamental pathological mechanisms underlying bone-related diseases. Therefore, it establishes a comprehensive therapeutic framework for the prevention and treatment of bone-related diseases while minimizing off-target effects.

Figure 5.

Therapeutic strategies targeting the gut-immune-bone axis.

Current therapeutic approaches focus on ameliorating gut dysbiosis in patients with bone-related diseases through interventions including probiotics, prebiotics, FMT, dietary modifications, and pharmacological agents. Restored microbial homeostasis facilitates immunomodulatory reprogramming of critical immune cell populations (Treg, Breg, macrophages), thereby rebalancing bone formation and bone resorption. The innovative therapeutic approach, characterized by its precise targeting of the gut-immune-bone axis, is of substantial significance in the context of both preventing and treating bone-related diseases. Created with BioRender.com

Prebiotics and probiotics

Probiotics, as key regulators of gut microbiota balance, recalibrate host physiological functions through various mechanisms, including competitive colonization, metabolic byproduct secretion, and immunomodulation. Prebiotics, on the other hand, are nutrient substrates that are specific to certain microbial taxa, capable of selectively enriching beneficial microbial communities.²⁷⁴ Together, they establish a tight bridge for “microbe-host” interactions.²⁷⁵ Previous studies have predominantly concentrated on the direct modulation of the gut-bone axis in ameliorating bone pathologies.²⁷⁶ For example, two GABA-producing probiotics, Streptococcus thermophilus and Lactobacillus pentosus, along with GABA itself, have been shown to directly promote chondrocyte proliferation, inhibit inflammatory responses, and suppress cartilage degradation, thereby ameliorating OA.²⁷⁷ Furthermore, Lactobacillus plantarum GKM3 and Lactobacillus paracasei GKS6 not only enhanced OBs differentiation by increasing the expression of OBs marker genes such as BMP-2, but also downregulated the expression of RANK as well as c-Fos and inhibited the expression of the OCs-related gene TRAP, thus suppressing OCs formation.²⁷⁸ However, emerging research evidence suggests that probiotics and prebiotics also function through a sophisticated communication network of the gut-immune-bone axis, thereby contributing to bone health maintenance and playing a significant role in the therapeutic management of bone-related diseases.

Dysregulation of Treg/Th17 balance is not only a precursor to most immune homeostasis disturbances, but also a crucial factor in bone homeostasis imbalance. It has been demonstrated that the probiotic P. histicola could alleviate RA by enhancing the production of Treg cells and IL-10 while inhibiting antigen-specific Th17 cell responses.²⁷⁹ Similarly, LGG promoted the secretion of IL-10 and TGF-β by Treg cells and inhibited the secretion of IL-17/TNF-α mediated by Th17 cells, thereby remodeling the immune microenvironment and alleviating bone loss in OVX mice.²³³ It is noteworthy that the bidirectional regulation of the Treg/Th17 balance by prebiotics and probiotics exhibits heterogeneity. Studies have revealed that Lactobacillus casei CCFM1074 ameliorated arthritis in CIA rats by promoting the production of SCFAs, particularly butyrate, thereby upregulating the Treg/Th17 ratio. In contrast, Lactobacillus casei CCFM1075 possessed the capability to reduce the number of Th17 cells.²⁸⁰ Similarly, probiotics such as F. prausnitzii, B. longum RAPO, and S. boulardii primarily could treat RA by inhibiting Th17 cell differentiation.^44,281 Interestingly, S. boulardii enhanced joint repair in CIA rats by reducing Th17 cell proliferation, which in turn reduced the expression of MMPs and RANKL. The underlying mechanism involves the ability of S. boulardii to reduce the abundance of Firmicutes and Patescibacteria, thereby inhibiting the TLR2 signaling pathway.²⁰¹

As mentioned above, the gut microbiota primarily exerts its function by secreting metabolites; thus, metabolites are equally beneficial in the treatment of bone-related diseases when supplemented directly as prebiotics.^187,282 For instance, the supplementation of butyrate could increase levels of 5-HIAA, which subsequently activates Breg cells via AhR. This activation could promote IL-10 secretion, suppress inflammatory responses, and ameliorate arthritis in CIA mice.¹²² Furthermore, the supplementation of tryptophan metabolites, specifically IAA and IPA, has been shown to augment the population of M2 macrophages in the intestinal lamina propria. This process stimulated the secretion of significant quantities of IL-10, thereby facilitating osteogenesis while concurrently inhibiting osteoclastogenesis. These effects collectively contribute to enhancing bone mineral density and mass in OVX mice, suggesting a potential therapeutic strategy for OP.⁷⁴

In addition, the collaborative effects of prebiotics and probiotics provide a dual-layered guarantee in the therapeutic approach to bone-related diseases. It has been demonstrated that oral administration of P. distasonis could effectively alleviate the symptoms of CIA, including the reduction of joint inflammation and bone erosion. This efficacy was attributed to the ability of P. distasonis to promote the production of secondary bile acids such as DCA by the gut microbiota, which in turn activated the TGR5 signaling pathway. This activation facilitated macrophage polarization toward the M2 phenotype and inhibited the differentiation of Th17 cells. Notably, the synergistic effect of the prebiotic ginsenoside Rg2 enhanced the ability of P. distasonis to ameliorate RA symptoms.²⁶⁰ These findings suggest that the polarization balance between M1 and M2 macrophages is also regulated by the prebiotics and probiotics for bone homeostasis. The inhibition of bone resorption by the probiotic Lactobacillus casei was similarly associated with a decrease in M1 macrophages and an increase in M2 macrophages.²⁵⁹ Mechanistically, the prebiotic 2’-fucosyllactose could regulate the TLR4/MyD88/NF-κB signaling pathway, thereby reducing the M1/M2 ratio and promoting bone formation to ameliorate OP.²⁸³ Furthermore, the probiotic Bifidobacterium longum could enhance the differentiation of Breg cells in the bone marrow as well as in the spleen, and increase their production of IL-10, which significantly inhibits RANKL-induced osteoclastogenesis and enhances bone mineral density in OVX mice.²⁵⁰

FMT

FMT, a therapeutic strategy that involves the transfer of gut microbiota from a healthy donor to patients to restore the gut microecosystem, represents a breakthrough in microbiome engineering.²⁸⁴ This approach provides a transformative tool for addressing the systemic regulatory challenges of bone-related diseases. Recent studies have shown that FMT could be deeply integrated into the multidimensional regulatory network of the gut-immune-bone axis.²⁸⁵ Through the reconstruction of gut microbiota composition, reprogramming microbial metabolism, and remodeling immune homeostasis, FMT could reestablish the dynamic balance of bone metabolism. This advance provides a novel avenue for precise interventions in bone-related diseases.

FMT could mitigate the detrimental effects of Treg/Th17 imbalance on bone health by modulating the composition and metabolic activities of the gut microbiota. FMT has been shown to increase the richness of beneficial bacterial families, including Prevotellaceae, Lachnospirillaceae, and Ruminococcaceae, thereby promoting the production of elevated levels of SCFAs. These SCFAs play a crucial role in inhibiting OCs formation, effectively alleviating OVX-induced bone loss.²⁸⁶ Specifically, SCFAs could enhance the proliferation and function of Treg cells by inhibiting HDACs, leading to inhibition of osteoclastogenesis and prevention of bone loss.²³⁴ Similarly, FMT restored the balance of the gut microbiota, thereby enhancing GLCA production. Further studies have shown that GLCA could activate CAR and promote the differentiation of Treg cells, thereby reducing pro-inflammatory cytokines and increasing anti-inflammatory factors. Additionally, the increase in Treg cells could further enhance the osteogenic differentiation of bone marrow mesenchymal stem cells, thus alleviating OP.⁹² Moreover, FMT could restore gut microbiota homeostasis by increasing the abundance of Firmicutes and Proteobacteria, thereby promoting the production of tryptophan metabolites such as IAA.²⁸⁷ These tryptophan metabolites could activate AhR to regulate the Treg/Th17 balance and ameliorate diseases progression of CIA rats.⁹⁴ The above studies have shown that FMT exerts a precise regulation on Treg/Th17 balance by ameliorating the composition and metabolism of the gut microbiota, thereby improving bone-related diseases. However, emerging evidence suggested that FMT may be less effective in restoring the microbial composition of the small intestine and could potentially induce unintended metabolic and immunological alterations. In comparison to jejunal microbiota transplantation (JMT), FMT demonstrated higher colonization efficiency in the colon and cecum but exhibited reduced efficacy in engrafting within the small intestine. This discrepancy may be attributed to the distinct ecological conditions of the small intestine, which are more conducive to the proliferation of aerobic bacteria. Importantly, JMT predominantly modulated host metabolic pathways, including fatty acid synthesis and bile acid metabolism, whereas FMT primarily impacted immune-related pathways such as inflammatory responses and immune homeostasis. Furthermore, clinical studies have corroborated findings from preclinical mouse models, demonstrating that FMT can elevate SATB2 expression levels in the small intestine. This upregulation implies a potential transformation of small intestinal tissue characteristics toward a colonic phenotype.²⁸⁸ Consequently, for precision-targeted interventions involving the intestinal microbiota, region-specific and individualized therapeutic strategies should be developed to prevent maladaptive interactions between the microbiota and the host’s physiological environment. Further comprehensive investigations are warranted to evaluate both the short- and long-term consequences of FMT and to advance the development of more efficacious microbiota-based therapies.

Diet

Dietary patterns play a pivotal role in shaping the composition, diversity and function of gut microbiota, with distinct nutritional regimens exerting profound modulatory effects on microbial community stability, functional capacity, and ecological diversity within the gut.²⁸⁹ Therefore, choosing the appropriate diet is of significant importance for regulating the gut-immune-bone axis network to prevent and treat bone-related diseases.

Individuals with high dietary fiber intake showed significantly more beneficial bacteria in their gut microbiota. According to clinical studies, it has been confirmed that dietary fiber could promote the production of SCFAs in the gut microbiota of RA patients, increase the population of Treg cells, and decrease the population of Th17 cells, thereby regulating inflammatory responses and mitigating the progression of RA.²⁹⁰ It is worth noting that different fiber components exert distinct regulatory effects on the gut-immune-bone axis. The pectin- and inulin-rich diet could enhance butyrate production by the gut microbiota, activate the AMPK/STAT3 signaling pathway, and reverse the Treg/Th17 ratio in the joints of CIA mice.⁸⁸ Besides, a resistant starch-rich HFD could significantly suppress the progression of bone-related diseases, accompanied by the enrichment of Lactobacillus spp and Bifidobacterium spp. Concurrently, as the gut microbiota changes, SCFAs such as acetate, propionate, and isobutyrate in resistant starch-rich HFD mice increased significantly, and further promoted IL10 + Treg differentiation via GPR43-dependent pathway and inhibited OC activity through inhibition of HDAC activity.^291,292 Interestingly, certain dietary components traditionally perceived as unhealthy dietary have been found to possess bone-protective properties when consumed at specific dosages. Moderate alcohol consumption could increase the abundance of Muribaculaceae, whose metabolite acetate activates GPR43 to inhibit endoplasmic reticulum stress and inositol-requiring enzyme 1 phosphorylation in neutrophils. This process reduced the formation of neutrophil extracellular traps and significantly alleviated joint erosion in CIA mice.²⁹³ Furthermore, a high-magnesium diet could enrich the gut microbiota with Akkermansia, resulting in an increased number of Foxp3⁺ Treg cells and elevated production of IL-10, thus inhibiting the inflammatory response of RA. This effect has been confirmed to be microbiota-dependent through FMT experiments.²⁹⁴

Drugs

Current therapeutic strategies for bone-related disorders predominantly rely on pharmacological interventions, yet conventional drug therapies persistently encounter the dual challenges of off-target effects and insufficient drug accumulation in target organs.^295,296 Emerging evidence suggests that innovative approaches targeting gut microbiota modulation through the gut-immune-bone axis regulatory network may synergistically enhance drug therapeutic efficacy while mitigating drug-induced adverse events, thereby establishing a novel framework for optimized intervention in bone-related diseases.

MTX is considered as the primary treatment for RA, and its efficacy goes beyond mere immunosuppression.²⁹⁷ Nevertheless, clinical studies have demonstrated that patients with early-stage RA treated with a three-month regimen of MTX combined with GCs showed a significant increase in gut microbiota α-diversity, along with a reduction in synovial inflammation. Particularly, the elevated abundance of Blautia gnavus was strongly associated with restoration of the Treg/Th17 balance.¹⁸ These findings suggest that the therapeutic benefits of MTX are not limited to direct immunosuppressive effects. Conversely, its positive influence on the gut microbiota provides promising direction for optimizing MTX therapeutic strategies. Furthermore, traditional Chinese medicine, known for its minimal side effects, has demonstrated distinct advantages in the modulation of the gut-immune-bone axis for the treatment of RA. One typical example is sinomenine, which could facilitate the proliferation of Lactobacillus, thereby increasing the production of indole-derived metabolites (IA/IAA/IPA) and remodeling the Treg/Th17 imbalance via the AhR/STAT3 signaling axis.⁹⁴ Asiaticoside could function by increasing the population of butyrate-producing bacteria, which subsequently activates the GPR43-PPARγ pathway to facilitate the differentiation of IL-10⁺ Treg cells.²⁹⁸ These findings suggest that various drugs may achieve immune homeostatic remodeling through different pathways of gut microbiota restoration, ultimately achieving therapeutic goals for bone-related diseases. Besides, shepherd purse extract has been shown to enhance OBs differentiation and prevent bone loss by ameliorating gut microbiota imbalance as well as activating the BMP/TGF-β/Wnt signaling pathway.²⁹⁹ The opioid receptor antagonist naltrexone inhibited OCs differentiation, thereby alleviating bone and cartilage erosion. This effect was achieved through the suppression of the TLR4/NF-κB signaling pathway, remodeling the Treg/Th17 imbalance, and reducing RANKL expression in synovial tissue.³⁰⁰ Although some studies have not directly demonstrated that drugs interfere with the gut-immune-bone axis, it is undeniable that drugs may affect the gut microbiota homeostasis. The metabolites of gut microbiota are inevitably involved in the impactingthe host immune system, and in turn participate in the regulation of bone homeostasis.

In conclusion, targeting the gut microbiota via strategies such as probiotic/prebiotic supplementation, FMT, dietary modifications, and pharmacological interventions can exert immunomodulatory effects, providing an innovative approach for the treatment of bone-related diseases. However, targeted research on the gut-immune-bone axis is still incomplete, with the majority of studies focusing solely on associations among them. The direct effects of specific microbial species and their metabolites require further validation through animal experiments and clinical studies. Moreover, the heterogeneity in gut microbiota responses due to factors such as age, diet, and genetics complicates the development of standardized intervention strategies. Developing predictive models based on the characteristics of patients’ gut microbiota to enable precise guidance (e.g., tailored probiotic combinations), in conjunction with dynamic monitoring technologies (e.g., gut chip sensors) for real-time efficacy evaluation, will promote the application of personalized therapies. Moreover, future studies should prioritize the elucidation of the underlying mechanisms of gut-immune-bone axis-based therapies. Clarifying their interactions may help address the current limitations in treatment and provide a scientific foundation for precision prevention and management strategies.

Conclusion and perspectives

Traditional studies on the gut-bone axis have predominantly concentrated on the direct effects of gut microbiota within the bone microenvironment. In contrast, the triangular network of gut-immune-bone axis introduces the immune system as a pivotal mediator, thereby enriching the understanding of bone homeostasis regulation through more intricate mechanistic insights. This perspective not only addresses previously neglected immune components in gut-bone interactions, but also provides novel therapeutic insights into osteoimmune diseases. Here, we comprehensively dissected the action network of the gut-immune-bone axis, elucidating how gut microbiota-derived signals regulate immune cell dynamics, such as Th17 cells, Treg cells, and macrophages, and subsequently impact bone homeostasis. Under pathological conditions, through the coordination of multiple organs, gut dysbiosis leads to immune dysfunction, which in turn disrupts bone homeostasis and predisposes individuals to bone-related diseases including OA, OP, and RA. The coordination of multiple systems provides novel clues for the development of innovative therapeutic strategies for bone-related diseases. Targeting immune intermediates could provide a more precise approach to maintain bone homeostasis than direct microbial modulation while minimizing systemic side effects.

Nevertheless, there still remain several limitations. The review synthesizes current evidence, while the precise molecular mechanisms, particularly how microbial metabolites spatially and temporally regulate immune-bone crosstalk remain incompletely resolved. Furthermore, most evidence derives from preclinical models or in vitro studies, which may not fully recapitulate human microbiome-host interactions. Besides, the promising microbiota-targeted therapies we discussed above are mainly in theoretical or preclinical stages. There are significant challenges in translating these approaches into clinically feasible treatments for bone-related diseases. For the next phase of research, we propose the following studies for the current limitations.

In future research endeavors, multi-omics integration approaches should be prioritized. This includes metagenomics for determining microbiota composition, metabolomics for examining microbially-derived metabolites, and single-cell transcriptomics for exploring immune cell signatures. Such integration aims to delineate causal microbial signatures, spatiotemporal metabolite dynamics, and host-microbe interactions, thereby identifying key regulatory nodes within the gut-immune-bone axis. However, the highly heterogeneous and dynamic nature of the gut microbiota poses a significant challenge, as its composition and function are co-regulated by genetic background, ethnic variation, dietary habits, disease status, and environmental factors. To address this complexity, there is a growing trend toward adopting multicenter, multiracial cohort designs to infer causality and elucidate the specific mechanistic roles of microbiota in the gut-immune-bone axis.

Despite substantial advancements have been made in gut microbiota research, the application of conclusions from animal studies to human must remain highly cautious due to given a scarcity of corresponding human research. The findings of numerous published studies on the gut microbiota are only valid under specific conditions, and replication by other research institutions often fails to reproduce the same outcomes. This issue is primarily attributed to the lack of complexity and adaptability in animal models. The repeated colonization and establishment of the gut microbiota in highly controlled laboratory settings result in the complete loss of microorganisms that have co-evolved naturally. Consequently, conducting human research and establishing a comprehensive human gut microbiota database has become particularly important. In response to this scenario, future studies should validate the reliability of animal experimental conclusions through large-scale collection and analysis of human gut microbiota samples, thereby further clarifying the causal relationships between gut microbiota changes and multiple influencing factors. Meanwhile, the application of emerging alternative technologies, including human organoids, organs-on-a-chip, and organoids-on-a-chip, can effectively address interspecies differences, thereby offering robust support for strengthening the credibility of research conclusions. Furthermore, the application of humanized mice serves as a solid bridge between scientific research and clinical practice, offering more clinically pertinent data for the study of the gut microbiota.

As research progresses, formulating individualized therapeutic strategies in the medical field has become an inevitable trend. Nevertheless, the treatment of bone-related diseases is currently plagued by limited medical resources, unclear pathogenic mechanisms, and substantial inter-individual variability in therapeutic outcomes. Therefore, the introduction of the gut-immune-bone axis concept facilitates the implementation of individualized therapeutic strategies that target the gut microbiota of patients with bone-related diseases. The implementation of individualized therapeutic strategies can reduce adverse effects, increase cure rates, and enhance the initiative of patients in disease prevention and treatment. This undoubtedly opens up new possibilities for the treatment of bone-related diseases and carries profound significance. However, due to issues such as the limited accuracy of gut microbiota sequencing technology, the large population of patients, and the absence of sufficient clinical research data, the implementation of individualized therapeutic strategies faces considerable challenges. To facilitate the development of more effective individualized therapeutic strategies, future research should establish a more comprehensive human gut microbiota database and perform more detailed classification of the gut microbiota, thereby enabling the identification of the specific characteristics of different gut microbial communities. Considering the multitude of factors that influence changes in the gut microbiota, the implementation of individualized therapeutic strategies necessitates comprehensive assessment to prevent the interference of interactions among individuals and between individuals and their environment on the accuracy of treatment. Furthermore, the establishment of stage-specific treatment objectives not only helps prevent adverse events but also enables timely assessment of the clinical treatment efficacy. The implementation of individualized therapeutic strategies offers promising insights for the adjunctive treatment of bone-related diseases and drives the progress of precision medicine along the gut-immune-bone axis.

Funding Statement

This work was supported by National Natural Science Foundation Regional Innovation and Development Joint Fund No. [U23A20413], National Natural Science Foundation of China No. [82172448], Key Clinical Cultivation Discipline Construction Project of PLA No. [145AHQ141009000X], Key Clinical Specialty of PLA No. [51561Z23711], Natural Science Foundation of Chongqing No. [cstc2021jcyj-msxmX1157], Young Elite Scientist Sponsorship Program by Association for Science and Technology of Qinghai Province No. [2024QHSKXRCTJ35] and Medical Innovation of Graduate Students in Chongqing No. [CYS23768].

Abbreviations

AhR

aryl hydrocarbon receptor;

AKT

protein kinase B;

AMPK, AMP-

protein kinase;

ACPA

anti-citrullinated protein antibodies;

BC

Bacillus coagulans;

BEVs

bacterial extracellular vesicles;

BMP

bone morphogenetic protein;

Breg

regulatory B;

BDMARDs

biologic DMARDs;

CAR

constitutive androstane receptor;

CCL, C-C

motif chemokine ligand;

CIA

collagen-induced arthritis;

CsDMARDs

conventional synthetic

DMARDs

CXCL, CXC chemokine ligand;

CXCR

CXC chemokine receptor;

DCA

deoxycholic acid;

DCs

dendritic cells;

DlaT

dihydrolipoamide acetyltransferase;

DMARDs

disease-modifying anti-rheumatic drugs;

EVs

extracellular vesicles;

FFAR

free fatty acid receptor;

FLS

fibroblast-like synoviocytes;

FMT

fecal microbiota transplantation;

Foxp

forkhead box P;

FXR

farnesoid X receptor;

GALT

gut-associated lymphoid tissues;

GCs

glucocorticoids;

GF

germ-free;

GLCA

glycochenodeoxycholic acid;

GPR

G-protein coupled receptor;

GrB

granzyme B;

HDAC

histone deacetylase;

HFD

high-fiber diet;

IA

indole acrylic acid;

IAA

indole-3-acetic acid;

IAld

indole-3-aldehyde;

ILCs

innate lymphoid cells;

IFN-I

type I interferon;

IGF-1

insulin-like growth factor 1;

JMT

jejunal microbiota transplantation;

L

murinus, Lactobacillus murinus;

LGG

Lactobacillus rhamnosus GG;

LCA

lithocholic acid;

LPS

lipopolysaccharide;

MAPK

mitogen-activated protein kinase; MHC Class II, major histocompatibility complex class II; MTX methotrexate;

NF-Κb

nuclear factor-kappa B;

NLRP

nucleotide-binding oligomerization domain-like receptor protein 3;

OA

osteoarthritis;

OMVs

outer membrane vesicles;

OP

osteoporosis;

OPG

osteoprotegerin;

OVX

ovariectomy;

PI3K

phosphoinositide 3-kinase;

PPARγ

peroxisome proliferator-activated receptor gamma;

PTH

parathyroid hormone;

RA

rheumatoid arthritis;

RANK

receptor activator of nuclear factor kappa-B;

RANKL

receptor activator of nuclear factor kappa-B ligand;

RF,

rheumatoid factor;

RORγt

retinoic acid receptor-related orphan nuclear receptor gamma t;

ROS,

reactive oxygen species;

SCFAs,

short-chain fatty acids;

scRNA-seq,

single-cell RNA sequencing;

SDI,

socio-demographic index;

SFB,

segmented filamentous bacterium;

SPF,

specific pathogen-free;

STAT,

signal transducer and activator of transcription;

TGR5

Takeda G protein-coupled receptor 5;

TGF-β

transforming growth factor-beta; Th, T helper;

Tfh,

T follicular helper;

THH,

Tripterygium hypoglaucum (Levl.) Hutch;

TPN,

total parenteral nutrition;

TLR,

toll-like receptor;

TMAO,

trimethylamine N-oxide;

TNF-α,

tumor necrosis factor-alpha;

TRAP,

tartrate-resistant acid phosphatase;

TREM,

trigger receptor expressed on myeloid cells;

Treg,

regulatory T;

VDR,

vitamin D receptor;

WMT

washing microbiota transplantation;

Wnt,

Wingless-related integration site;

YLD,

years lived with disability.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.