Role of Cytokines in Bone Diseases and Their Therapeutic Application

Introduction

Bone is a dynamic tissue composed of 60% crystalline hydroxyapatite (a mineral crystal), 30% organic matrix, and approximately 10% cells, and it continually remodels.¹ Bone remodeling includes bone resorption followed by bone formation. Bone cells within bone tissue govern this process. Osteoclasts are cells that attach to the matrix and resorb older bone using acidic enzymes. These are typically large, multinucleated cells derived from hematopoietic progenitor cells belonging to the myeloid-lymphoid lineage. Osteoblasts are cells that help form new bone; together with osteoclasts, they help heal damaged bone. Osteocytes, derived from osteoblasts, constitute more than 90% of the total bone cells. They remain embedded in the bone matrix and serve as the primary mechanosensor and regulator of bone mineral metabolism. The overall maintenance of bone homeostasis is achieved when both cell types function in a coordinated manner. Bone remodeling is critical for adapting to environmental changes and ultimately maintaining a delicate balance between bone renewal and repair. The imbalance between bone resorption and formation can lead to either too low or too high bone density, ultimately resulting in diseases such as osteoporosis (low bone density) and osteopetrosis (high bone density).² There are several metabolic bone diseases characterized by overall bone loss, including osteoporosis, osteomalacia, osteogenesis imperfecta, Paget’s disease of bone, and fibrous dysplasia. In contrast, patients with osteopetrosis develop an abnormally high bone density. Intriguingly, these changes in bone mass are accompanied by alterations in the cytokine profile.^1,3

Cytokine activity is not solely associated with skeletal disorders. Several cytokines play an important role in bone cell differentiation, even under normal, healthy conditions. The receptor activator of nuclear factor kappa B ligand (RANKL) is a cytokine that acts through its cognate receptor, RANK, or its decoy receptor, osteoprotegerin (OPG), to play a crucial role in bone homeostasis.⁴ RANKL is also known as tumor necrosis factor-related activation-induced cytokine (TNF-related activation-induced cytokine, TRANCE). TRANCE/RANKL knockout mice are known to be protected from bone resorption in the serum transfer model of arthritis.⁵ OPG^−/− mice develop early-onset osteoporosis and arterial calcification.⁶ Macrophage colony-stimulating factor (M-CSF, also known as colony-stimulating factor CSF-1) is a cytokine that binds to CSF-1R (c-Fms) on osteoclast precursor cells, promoting their differentiation, proliferation, and survival. OPG secreted by osteoblasts binds to RANKL, inhibits the RANKL-RANK interaction, suppresses the formation of osteoclasts, and typically acts as a negative regulator of bone damage.⁷ Thus, the RANKL/RANK/OPG axis is a prominent therapeutic target in skeletal disorders. Osteoblasts are the primary bone-forming cells that secrete collagen and non-collagenous proteins, osteopontin, and osteocalcin, which are essential for bone formation.⁸ In normal physiological conditions, the functions and differentiation of osteoblasts are regulated by cytokines such as transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α).⁹

However, despite their indispensable role in maintaining bone homeostasis, cytokine overactivity has been established as a key feature of the pathophysiology of various skeletal disorders, including rheumatoid arthritis, osteoarthritis, and osteoporosis.⁴ There are instances in which bacterial or viral infections can lead to the development of skeletal diseases and the degradation of bone mass. Organisms such as Streptococcus aureus, Porphyromonas gingivalis, and Chikungunya viruses invade bone cells and destroy osteoblasts, thereby contributing to joint pain and bone loss in diseases such as osteomyelitis, periodontitis, and chikungunya. Such diseases pose a threat to humanity, as the relevant pathogens can remain dormant for long periods and form distinct biofilms within tissues. As a result, antibiotics cannot eliminate them from the system.^10–13 The toxins released by these pathogens activate the immune system to produce pro-inflammatory cytokines, such as IL-1, TNF-α, and IL-6.^14–16 The overexpression of pro-inflammatory cytokines leads to the infiltration of immune cells, which further mount an inflammatory response.¹ Moreover, pro-inflammatory cytokines promote osteoclast differentiation, consequently enhancing bone damage.^17–19

Therefore, uncontrolled cytokine activity can lead to severe bone damage, especially in infection-mediated skeletal diseases. However, the pathomechanism of these diseases remains poorly understood. This review highlights the role of cytokines in regulating bone homeostasis, encompassing epigenetic and metabolic downstream mechanisms. Additionally, the review summarizes how cytokine-mediated bone remodeling contributes to worsening infectious disease-associated bone loss and how it has been targeted therapeutically to manage infection-mediated bone loss, despite pertinent limitations to this approach.

Role of Cytokines in Bone Remodeling

Cytokines play a crucial role in driving the differentiation and function of osteoclasts and osteoblasts. Consequently, these cytokines are also associated with bone resorption and bone formation and influence bone remodeling (Figure 1).

Figure 1 Effect of cytokines on bone remodeling. Immune cells, including dendritic cells, neutrophils, macrophages, T cells, B cells, monocytes, eosinophils, and basophils, produce cytokines that regulate bone remodeling. (A) TNF-α, IL-1, IL-8, IL-15, IL-17, IL-20, and IL-34 enhance osteoclast differentiation. The expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metalloproteinase-9 (MMP-9), and calcitonin receptors, increases, leading to bone damage. Osteocytes express RANKL, sclerostin, IL-1β, and TNF-α, which contribute to increased osteoclastogenesis and bone loss. IL-3, IL-4, IL-12, IL-13, IL-18, IL-23, IL-27, IL-29, IL-35, and IFN-γ are cytokines that decrease osteoclast differentiation and inhibit the expression of osteoclast-specific genes, thereby preserving healthy bone. (B) IL-3, IL-32, IL-35, and IFN-γ promote calcification and osteogenesis, supporting healthy bone formation. IL-10, IL-15, IL-17, and IL-27 induce osteoblast apoptosis and bone damage. Note: The green arrow indicates upregulation, and the red arrow indicates downregulation.

Effect of Cytokines on Osteoclast and Osteoblast Differentiation

Interleukin (IL)-1 Family Members

IL-1 family cytokines (IL-1α, IL-1β, IL-8, and IL-33) are actively expressed in inflammatory tissues and promote osteoclast differentiation.²⁰ IL-1 can also upregulate RANKL, a crucial factor in osteoclastogenesis. Overexpression of IL-1α or IL-1β, or deletion of IL-1R antagonist (IL-1Rα), causes the development of arthritis involving cartilage and bone erosion.²¹ The TNF-transgenic model of inflammatory arthritis revealed that blocking IL-1 protects mice from bone loss.^21,22 IL-1β indirectly enhances TNF-α-induced osteoclast differentiation. It promotes RANKL expression in stromal cells by activating the mitogen-activated protein kinase (MAPK) pathway. Apart from this, IL-1β also promotes osteoclastogenesis by increasing insulin-like growth factor 2 (IGF2) and chemokines (such as CXCL1, CXCL7, and stromal cell-derived factor 1) in non-osteoclasts.²³ Thus, this cytokine is expected to increase osteoclast differentiation.^23,24 In contrast to IL-1β, IL-18, another member of the IL-1 family, was observed to inhibit bone resorption. As this cytokine induces GM-CSF expression, it drives osteoclast precursors toward the dendritic cell lineage. IL-33 is also another potent suppressor of osteoclast differentiation. IL-33 cytokine promotes the differentiation of mononuclear osteoclast precursors into dendritic cells and macrophages, thereby impairing osteoclast development.²⁵

In line with previous findings, IL-1 can influence fracture healing by inhibiting human osteoblast migration. Additionally, IL-1 was found to activate p38 MAPK in osteoblasts, thereby inducing bone resorption. Osteoclasts produce IL-8, which enhances their differentiation in an autocrine manner. During oxidative stress, human osteoblast cells produce increased levels of IL-6, IL-8, and TNF-α, which can be inhibited by Luteolin (3′,4′,5,7-tetrahydroxyflavonone).²⁶ Osteoclastogenesis can be inhibited by adding anti-IL-8 antibodies, IL-8 receptor inhibitors, or the antioxidant luteolin. Inhibition of IL-8 helped control tooth decay and periodontitis. However, the detailed molecular mechanisms and functions of IL-8 in bone biology remain to be precisely elucidated.

IL-33 is a member of the IL-1 cytokine family that acts via the ST2 receptor. IL-33 inhibits osteoclast differentiation and plays a protective role in bone health.²⁷ This cytokine suppresses RANKL-induced NFATc1 activation by regulating the functions of Blimp-1 and interferon regulatory factor-8 (IRF-8).^28,29 Besides, IL-33 promotes osteoclast apoptosis by enhancing pro-apoptotic molecules, such as Bcl-2-associated X protein (BAX), Fas, Fas ligand (FasL), and Fas-associated death domain (FADD).³⁰ In vitro experiments with bone marrow cells revealed that IL-33 induces the expression of IL-4, IL-10, IL-13, and GM-CSF mRNA.³¹ In the in vivo model, IL-33 suppressed TNF-α-induced osteoclast formation and bone resorption.³² Reportedly, IL-33 expression was reduced in osteoporotic women compared with healthy controls.³¹ Mice deficient in ST2 receptors (IL-33R) show normal bone formation but increased osteoclast activity. Therefore, these mice exhibit a decreased trabecular bone mass.³³ Recently, it has been reported that IL-33-induced TERM2⁺ macrophages promote new bone formation in Ankylosing spondylitis.³⁴ Additionally, it was observed that ovariectomy-induced bone loss is IL-33/ST2-dependent, occurring in the maxilla but not in the femur, suggesting a bimodal, site-specific role of IL-33 in bone remodeling.^35,36

IL-33 also exerts simultaneous osteoprotective effects by promoting mineral deposition in the bone matrix and enhancing the expression of genes involved in osteoblast function. Additional studies have shown that it can maintain reduced sclerotin mRNA levels in ascorbate-treated primary osteoblasts for a prolonged period.²⁵ It has been reported that intracellular IL-33 in the osteoblast nucleus acts as a repressor of NF-kB signaling. In contrast, secreted extracellular IL-33 is known to promote osteoclastogenesis by increasing the RANKL expression in osteoblasts.³⁷ However, a detailed evaluation of the cellular and molecular mechanisms of IL-33, including the cell types and triggers involved in the extracellular versus nuclear expression, is strongly required. Moreover, the expression, localization, and contribution of IL-33 in bone remodeling need further investigation.

IL-37 belongs to the IL-1 cytokine family. However, IL-37 inhibits osteoclast differentiation and bone resorption.³⁸ It has been shown that IL-37 promotes the bone healing process in rat skulls by activating the PI3K/AKT pathway. Like other cytokines with osteoprotective roles, it can promote the differentiation of mesenchymal stem cells (MSCs) into osteoblasts by upregulating osteoblast-specific gene expression.³⁹ IL-37 also inhibits NLRP3 inflammasome activation, modulates M1/M2 macrophage polarization, and ameliorates periodontitis.⁴⁰ In a contradictory report, ankylosing spondylitis patients show elevated levels of IL-37; thus, this cytokine is hypothesized to remain associated with osteoporosis as well.^41,42

IL-3

IL-3 is a multipotent hematopoietic cytokine released by monocytes, macrophages, stromal cells, and activated T cells.⁴³ It significantly increases osteoblast differentiation from human mesenchymal cells and promotes matrix mineralization during bone formation. It has been reported that the JAK/STAT signaling pathway enhances BMP-2 secretion and promotes osteoblast differentiation.⁴⁴ On the other hand, IL-3 inhibits osteoclast differentiation.^45,46 One study demonstrated that IL-3 enhances RANKL mRNA and protein expression and also affects OPG expression. RANKL has two forms: membrane-bound and soluble. IL-3 regulates both forms of RANKL. IL-3 can downregulate the soluble form of RANKL by decreasing the levels of metalloproteases, such as MMP3, ADAM10, ADAM17, and ADAM19. It can also increase membrane-bound RANKL expression via the Akt2/STAT5 signaling pathway.⁴⁷

IL-2 Family Cytokines

Various IL-2 family cytokines play a prominent role in regulating bone remodeling. Interleukin-7 belongs to the IL-2 cytokine family and is released by stromal cells and osteoblasts in response to IL-1 or TNF-α.⁴⁸ While IL-7 is overexpressed in mice, osteoclast activity increases, leading to decreased bone mass.⁴⁹ IL-7 also induces T cells to express RANKL and TNF-α, thereby promoting osteoclast differentiation. Nude mice lacking T cells fail to show IL-7-driven bone loss.⁵⁰ Ovariectomized mice exhibit elevated levels of IL-7, which upregulate development and bone loss.⁵¹

Additionally, this cytokine helps in B-cell differentiation.⁵² IL-7 induces proliferation in B cells in rodents, while IL-7R-deficient mice are protected from bone loss.⁵³ According to recent reports, IL-7/IL-7R signaling activates the c-fos/c-jun pathways, thereby enhancing NFATc1, CTSK, and MMP-9 expression, leading to increased osteoclast activity and bone damage.⁵⁴ Moreover, IL-7 can directly promote osteoclast differentiation, independent of RANKL stimulation, by activating the JAK/STAT pathway.⁵⁵ Further, contrary to these reports, IL-7 was also observed to inhibit osteoclast differentiation in vitro. This effect was observed when bone marrow cells were cultured with IL-7, CSF-1, and RANKL.⁵⁶ In the periodontitis model, IL-7 induces osteoclast differentiation from fibroblast-driven macrophages.⁵⁷ This phenomenon suggests that targeting IL-7R may help in controlling bone resorption in periodontitis.

IL-15 is another cytokine belonging to the IL-2 superfamily. IL-15 synergizes with RANKL to promote osteoclast differentiation and activate the ERK pathway.⁵⁸ The mechanism of action of IL-15 is similar to that of IL-2. It also shares many similarities with IL-7.⁵⁸ To investigate the effect of IL-15 on osteoblasts, osteoblasts were co-cultured with purified natural killer (NK) cells. It has been found that higher IL-15 doses increased caspase-3 expression in NK cells, thereby enhancing osteoblast apoptosis.

IL-6

IL-6 was demonstrated to enhance osteoclast differentiation indirectly.⁵⁹ IL-6 upregulates RANKL expression in osteoblasts, thereby increasing osteoclast differentiation through interaction with mesenchymal stem cells.^60,61 However, IL-6 and its soluble receptor, IL-6R, promote osteoclastogenesis by triggering trans-signaling through the adaptor protein gp130.⁶² Neutralizing IL-6R inhibits osteoclast formation and blocks the progression of bone damage in patients with rheumatoid arthritis (RA). Like TNF-α, IL-6 also makes a dual contribution to the osteogenesis process. It can affect osteoclast and osteoblast formation based on the stress to which mice have been exposed. It has been reported that, after ovariectomy, IL-6^−/− mice exhibit increased expression of genes involved in osteoblast differentiation, including Col1a1 and Runx2. Interestingly, these mice show a decreased expression of osteoclast-related genes, including cathepsin K (CTSK), MMP9, and tartrate-resistant acid phosphatase (TRAP). Some studies also suggest that the IL-6/IL-6R complex can enhance the differentiation of BM-MSCs (bone marrow-derived mesenchymal stem cells) through a paracrine/autocrine feedback loop and by directly activating the downstream STAT3 signaling pathway.⁶³

Oncostatin M (OSM) is a member of the IL-6 family and is released at all stages of osteoblast differentiation.^64,65 OSM induces stromal cells to express RANKL, thereby increasing osteoclast differentiation.^66,67 OSM acts via OSMR to stimulate RANKL expression and osteoclast formation.⁶⁴ Interestingly, elevated serum IL-6, along with TGF-β, has been reported to show promise as a diagnostic biomarker in elderly male patients with osteoporosis.⁶⁸

IL-10 Family Cytokines

IL-10 is a cytokine released by Treg cells, macrophages, and B cells, and it is a potent inhibitor of osteoclast differentiation. IL-10 inhibits osteoclast formation through the RANK/RANKL/OPG axis.⁶⁹ IL-10 expression levels decrease in osteoporosis, both in mice and humans,^70,71 but this expression is restored after anti-osteoporotic treatment.⁷² IL-10 acts on the osteoclast precursor cells to inhibit the formation of osteoclasts at the early stage of differentiation, and in this process, IL-10 enhances the expression of OPG.^73,74 IL-10 inhibits calcium signaling downstream of RANK/RANKL interaction, thus inhibiting RANKL-induced osteoclast differentiation. IL-10 also suppresses the expression of c-fos and c-jun during osteoclast formation.^75,76 In ovariectomized mice, the number of IL-10-producing B cells decreases, whereas the frequency of inflammatory Th17 cells increases; when IL-10-producing B cells were adoptively transferred, they protected against osteoporosis.

Osteoporotic patients have lower levels of IL-10 than healthy individuals.⁷⁰ In a report, IL-10 suppressed the synthesis of bone-related proteins, including alkaline phosphatase (ALP), osteocalcin, and type 1 collagen. Additionally, IL-10-treated stromal exhibited a mineralized extracellular matrix compared to the control condition, suggesting that IL-10 regulates osteogenic differentiation. This study also showed that IL-10 can inhibit bone mineralization in mouse bone marrow cells, thereby supporting its inhibitory role in osteogenic differentiation and bone formation.⁷⁷

IL-19 is an anti-inflammatory cytokine that belongs to the IL-10 family of cytokines. IL-19 inhibits osteoclast formation by suppressing RANKL-induced NF-κB and p38MAPK pathway.⁷⁸ In contrast, IL-19 promotes the production of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which, in turn, increase RANKL expression in synovial fibroblasts, thereby enhancing osteoclast differentiation and bone damage in arthritis.

IL-20 is another member of the IL-10 cytokine family. IL-20 had a higher serum concentration in patients with osteoporosis. The application of anti-IL-20 monoclonal antibodies inhibits M-CSF and RANKL-mediated osteoclast differentiation.⁷⁹ IL-20 promotes osteoclast formation by upregulating NFATc1, NF-κB, STAT3, TRAF6, and c-fos. IL-20 also increases soluble RANKL in osteoblasts.^79,80 The anti-IL-20 antibody could be a promising therapeutic option for patients with osteoporosis.⁸⁰

Mouse models, such as ovariectomized (OVX) mice, also show similar results. In OVX mice, IL-20 is significantly upregulated in serum. IL-20 alters the expression of transcription factors, thereby preventing osteoblast differentiation and survival. It increases sclerostin expression and decreases those of osterix, osteoprotegerin (OPG), and runt-related transcription factor 2 (RUNX2).⁸⁰ Another study demonstrated that IL-20 acts in an autocrine manner to upregulate RANKL expression in osteoblasts, thereby affecting osteoblast differentiation.⁷⁹

IL-11

IL-11 (also known as adipogenesis inhibitory factor, AGIF), produced by bone marrow stromal cells and osteoblasts, is a novel multifunctional cytokine whose role in bone remodeling is not fully understood.⁸¹ IL-11 has been reported to influence stromal cell hematopoiesis by regulating adipocyte differentiation and inhibiting adipogenesis in the bone marrow microenvironment.⁸² The role of the IL-11/IL-11Rα signaling axis in osteoblast progenitor cells was also explored to ensure their development and survival.^81,83 Interestingly, IL-11 stimulated osteogenesis in addition to its pro-osteoclastic effects.⁸⁴ IL-11 was shown to promote the differentiation of pluripotent mesenchymal progenitors towards the osteoblast lineage. IL-11 overexpression in transgenic mice increased bone formation, enhancing cortical thickness and the strength of long bones.⁸⁵ It has been demonstrated that mechanical stress on bone stimulates IL-11 expression, which, in turn, promotes osteoblast differentiation.⁸⁶

On the other hand, IL-11Rα deficiency led to defects in bone remodeling, including reduced bone resorption, increased trabecular mass, and enhanced systemic adiposity.^87,88 IL-11 also regulates several pathways related to senescence and aging.^89,90 These findings highlight the therapeutic significance of IL-11 in addressing diseases characterized by dysregulated bone resorption and osteogenesis, including osteoporosis and rheumatoid arthritis.

IL-12 Family of Cytokines

The IL-12 family of cytokines comprises IL-12, IL-23, IL-27, and IL-35 and plays a very important role in shaping the immune response and bone physiology through heterodimeric subunits and a distinct JAK-STAT signaling pathway.⁹¹ IL-12 exhibits an anti-osteoclastic function, inhibiting RANKL-induced osteoclast differentiation by suppressing NFATc1.⁹² IL-12 also induces apoptosis in osteoblasts by recruiting the Fas/FasL pathway.⁹³ IL-12 synergizes with IL-18 to induce apoptosis in osteoclasts as well. However, the function of IL-12 in animal models remains poorly understood. IL-12 has a dual effect on the activity of both osteoblasts and osteoclasts. It can induce osteoblast apoptosis via the FAS/FASL pathway, thereby inhibiting osteoblast differentiation and bone formation.⁹³

IL-23 plays a crucial role in the proliferation of Th17 cells and is implicated in several autoimmune diseases.⁹⁴ IL-23-deficient mice are protected from collagen-induced arthritis, and it is also known to promote osteoclastogenesis by inducing RANKL in myeloid precursor cells.⁹⁵ Mice deficient in IL-23 expression were protected from bone loss.⁹⁴ IL-23 enhances osteoclast differentiation by promoting RANKL expression in bone marrow cells⁹⁵ and CD4⁺ T cells.⁹⁶ It has been observed that patients undergoing long-term treatment with anti-IL-23-based biologics experience temporary bone loss. Additionally, IL-23R^−/− mice exhibit a transient defect in bone mass.⁹⁷

IL-27 exploits IL-27R and the gp130 complex to exert its function. IL-27 is an anti-osteoclastic factor that inhibits RANKL-induced activation of the MAPK and NF-κB pathways and downregulates NFATc1 and c-fos expression.⁹⁸ IL-27 may suppress osteoclast differentiation by inhibiting STAT1-mediated c-fos expression.⁹⁹ Additionally, IL-27 inhibits the STAT3-mediated expression of both membrane-bound and soluble RANKL in CD4⁺ T cells.¹⁰⁰ In ovariectomized mice, IL-27 decreases Th17 differentiation by suppressing RORγt expression, increases IL-10 to promote Treg differentiation¹⁰¹ and protects bone. Therefore, IL-27 can be considered a potential therapeutic agent for the treatment of osteoporosis. Reports have recently shown that IL-27 can affect osteoblast and osteoclast differentiation by regulating the transcription factor Egr-2 (early growth response-2). When OVX mice were treated with IL-27, region-specific bone parameters were affected. In the cortical region, bone preservation was observed.

In contrast, a trabecular bone loss was observed.¹⁰¹ The preservation of cortical bone parameters was later found to be associated with decreased Th17 cell differentiation and increased Treg cell production. Treg cells upregulate anti-apoptotic factors (eg, MCL-1) in osteoblasts, thereby preventing their apoptosis.¹⁰¹

IL-35 is a potent anti-inflammatory and immunosuppressive factor.¹⁰² IL-35 directly inhibits osteoclast differentiation by suppressing TNF-α-induced NFATc1, c-fos, NF-κB, and MAPK activation. Additionally, IL-35 activates the JAK1/STAT1 pathway, leading to apoptosis.¹⁰² IL-35 promotes MSC proliferation while blocking their adipogenic potential. IL-35 enhances the expression of β-catenin and axin-2.¹⁰³ IL-35 can inhibit RANKL- and M-CSF-induced osteoclast differentiation triggered by the Th17/IL-17 axis and can control collagen-induced arthritis in a mouse model, suggesting it could be a promising therapeutic target for osteoporosis.^104,105 It promotes MSC differentiation into osteoblasts by upregulating the expression of β-catenin and Axin2, key players in the Wnt/β-catenin/PPARγ pathway.¹⁰⁶ It balances the differentiation of progenitor cells into lipogenic or osteogenic lineages, suggesting its therapeutic potential in osteoporosis and obesity.¹⁰⁴

IL-13

IL-13 is an anti-osteoclast cytokine secreted by Th2 cells. The biological functions of IL-13 are similar to those of IL-4. IL-13 induces endothelial cells to produce osteoprotegerin (OPG), a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL). The binding of OPG with RANKL blocks osteoclast formation. STAT6 is activated downstream for the release of OPG.¹⁰⁷ Additionally, IL-13 and IL-4 can inhibit cyclooxygenase-dependent prostaglandin secretion by osteoblasts, thereby suppressing osteoclast differentiation and bone loss.¹⁰⁸ One study demonstrated that IL-13 inhibits IL-1α-induced bone-resorbing activity in mice.¹⁰⁸ IL-13 and IL-4 inhibit COX-2 (cyclooxygenase-2)-dependent prostaglandin synthesis, thereby further inhibiting bone resorption.¹⁰⁸

IL-17

IL-17 can be expressed by mast cells in the inflamed synovial tissue of arthritis. IL-17 has been shown to promote osteoclast differentiation by stimulating RANKL expression in mesenchymal cells.^109,110 The number of Th17 cells was elevated in ovariectomized mice and in postmenopausal women, along with the secretion of IL-17A.¹¹¹ Enhanced IL-17, in turn, increases the production of other pro-inflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-8, and RANKL, in osteoblasts.^112,113 Additionally, IL-17 induces autophagy in osteoclast precursors and supports osteoclast differentiation by activating the JNK pathway.¹¹⁴ In an animal model of inflammatory arthritis, blocking IL-17 protected against bone loss. Estrogen treatment and anti-IL-17 agents can control post-ovariectomy bone loss and promote new bone formation by increasing FOXO1 and ATF4 activity.¹¹⁵ However, the function of IL-17 varied with concentration. A higher concentration of IL-17 inhibited osteoclast differentiation from RAW 264.7 cells and suppressed bone resorption by downregulating the expression of CTSK and MMP-9. Alternatively, IL-17 promotes the secretion of chemokines, including CXCL1/KC/GROa, CXCL2/MIP2a/GROb, CXCL8/IL-8, CCL2/MCP1, and CCL20/MIP3a, from bone cells, cartilage, synoviocytes, and macrophages. It can also drive the recruitment of neutrophils, macrophages, and T cells to the inflamed synovium.¹ Secukinumab, an anti-IL-17 antibody, reduces clinical pathophysiology in ankylosing spondylitis and rheumatoid arthritis.

IL-17 activates the MEK/ERK signaling pathway, which generates reactive oxygen species, to induce human mesenchymal stem cells. These pathways also contribute to the proliferation, migration, and differentiation of osteoblasts from human mesenchymal stem cells.¹¹⁶

IL-17, like many other cytokines, has a dual effect on osteoblast differentiation. It can positively regulate osteoblast differentiation from precursors.¹¹⁷ In contrast, IL-17 has also been shown to inhibit bone regeneration and osteoblast differentiation in rodents.¹¹⁸ One report shows that IL-17 can stimulate osteoblast secretion at high concentrations. Osteoblasts secrete factors that activate the NLRP3 inflammasome, thereby enhancing RANKL and IL-1 production, leading to severe disruption of bone metabolism.¹¹⁹ Additionally, IL-17 induces the expression of M-CSF and RANKL.

IL-18

IL-18 is a pleiotropic pro-inflammatory cytokine. However, IL-18 inhibits TNF-α-induced osteoclast differentiation by activating the Fas/FasL pathway, which induces apoptosis in osteoclast precursors.^120,121 However, anti-FasL antibodies failed to completely inhibit apoptosis. In the presence of TNF-α, IL-18 induces nitric oxide production, leading to the apoptosis of myeloid precursors.¹²² IL-18 promotes IFN-γ and GM-CSF production in T cells, ultimately inhibiting osteoclast formation.^123,124 Interestingly, IL-18 binding protein prevented bone damage in ovariectomized mice and osteoporotic patients.¹²⁵

IL-29

IL-29 is a member of the IFN cytokine family that shares the IL-28R1/IL-10R2 receptor complex and activates the JAK/STAT pathway downstream.¹²⁶ Dendritic cell-derived IL-29 inhibits osteoclast differentiation and bone resorption.¹²⁷ IL-29 suppresses RANKL-induced osteoclast differentiation by downregulating NFATc1 and NF-κB activation.¹²⁸

IL-32

IL-32 is a pro-inflammatory cytokine. IL-32 has seven isoforms: α, β, γ, δ, ε, θ, and ζ.¹²⁹ It has been reported that IL-32α and IL-32β/γ isoforms have opposing functions in promoting monocyte-derived osteoblast/osteoclast differentiation in patients with HIV infection.¹³⁰ Mice overexpressing IL-32 exhibit enhanced osteogenic potential and bone formation with age.¹³¹ These mice were protected from osteoporosis-induced bone loss by upregulation of miR-29.¹³¹ In humans, individuals with low serum IL-32γ levels have lower BMD.¹³¹ This phenomenon suggests that IL-32γ may have a protective role in preventing bone loss. The expression of each IL-32 isoform in various bone cells warrants detailed investigation, and its association with bone disorders remains to be evaluated.

IL-34

IL-34 is a proinflammatory cytokine that binds to four distinct receptors: CSF-1R, syndecan-1, PTP-z, and TERM.¹³² Human IL-34 exhibits approximately 72% and 71% identity with rat and mouse IL-34, respectively.¹³³ IL-34 can effectively compensate for M-CSF in osteoclast differentiation. Therefore, combined with RANKL, IL-34 can augment bone resorption. Administration of IL-34 in mice resulted in a loss of trabecular bone mass.¹³⁴ Injection of an anti-IL-34 neutralizing monoclonal antibody significantly reduced alveolar bone loss and the number of TRAP⁺ osteoclasts in periodontitis lesions.¹³⁵ IL-34 enhances the proliferation of bone marrow macrophages, thereby facilitating osteoclast formation by activating NFATc1 expression.¹³⁶ In contrast, an in vivo study suggests that low-dose IL-34 regulates osteogenesis in human bone marrow stem cells and promotes fracture healing by activating the PI3K/AKT and ERK pathways.¹³⁷

Interferons (IFNs)

IFNs can be classified into three main types: IFN-α, IFN-β, and IFN-γ. IFN-α and IFN-β inhibit RANKL-induced osteoclast differentiation by reducing the activation of c-fos.^138,139 It was observed that IFN-β suppressed osteoclast differentiation by enhancing nitric oxide (NO) release and inducing nitric oxide synthase (iNOS).¹⁴⁰ In vitro experiments have demonstrated that IFN-γ inhibits M-CSF- and RANKL-induced osteoclast formation by reducing NFATc1 expression.¹⁴¹ IFN-γ treatment degrades TRAF6 via the ubiquitin-proteasome system and, in turn, inhibits downstream JNK and NF-κB.^142,143 IFN-γ can induce osteoclast apoptosis by activating the Fas/FasL pathway.¹⁴⁴

Additionally, IFN-γ restricts osteoclast fusion by inducing guanylate-binding proteins.¹⁴⁵ In in vivo conditions, mice deficient in IFN-γR1 exhibit reduced bone mass after ovariectomy compared with wild-type mice. This phenomenon again confirms that IFN-γ is a crucial factor in bone formation and can also prevent bone resorption.¹⁴⁶ On the contrary, a study found that IFN-γ promoted osteoclast differentiation by enhancing fusion during osteoclast formation, and upregulating the dendritic cell-specific transmembrane protein (DC-STAMP).¹⁴⁷

Tumor Necrosis Factor Alpha (TNF-α)

TNF-α is a pro-inflammatory cytokine that induces osteoclastogenesis by acting directly and indirectly on osteoclast precursors.^148–150 TNF-α plays a crucial role in enhancing bone resorption, synergizing with RANKL. It was also observed that TNF-α induces osteoclast differentiation in arthritic joints by upregulating the expression of OSCAR on osteoclasts and their precursors. Clinical trials with anti-TNF-α neutralizing antibodies or soluble receptors, such as adalimumab, certolizumab, golimumab, and infliximab, have shown efficacy in RA patients by subsiding pain and joint inflammation. TNF-α induces NF-κB and PI3K/AKT signaling to enhance osteoclast formation in vitro.¹⁵¹

Additionally, TNFα has been shown to exert dose-dependent, bidirectional effects on osteoblast function and bone formation. TNF-α at its high concentration inhibits bone formation and osteoblast function, whereas in its low concentration, it induces mesenchymal cells to differentiate into osteoblasts.¹⁵² Reportedly, TNF-α inhibits the expression of genes that regulate osteoblast differentiation. For example, it can suppress osteoblast differentiation by inhibiting expression of RUNX2.¹⁵³ In the early stages of osteoblast formation from precursor cells, TNF-α can inhibit IGF-1 (insulin-like growth factor-1) expression, consequently suppressing osteoblast differentiation.¹⁵⁴ Recently, it has been reported that inflammatory osteoclasts drive colitis in a TNF-α-dependent manner by dysregulating myeloid differentiation.¹⁵⁵

None of these cytokines operates independently. Pro-inflammatory cytokines can synergize to amplify inflammatory responses several-fold. For example, Th1 cytokines, IFN-γ and TNF-α, can synergize in the context of airway inflammation and hyperresponsiveness.¹⁵⁶ IL-17A combined with TNF-α or IL-1 leads to even more increased inflammation.¹⁵⁷ IL-1β synergizes with IL-6, IL-21, IL-23, and TGF-β to promote the differentiation of naïve CD4⁺ Th cells into Th17 cells. There is ample evidence that pro-inflammatory cytokines can contribute to the induction of inflammation and bone damage in various skeletal diseases through both independent and combined effects.

Effect of Cytokines on Osteocytes

Osteocytes are a crucial cell type that serves as a bridge of communication between osteoclasts and osteoblasts.¹⁵⁸ The osteoblasts differentiate into osteocytes and become deeply embedded within the bone matrix. The functions of osteocytes are significantly affected by cytokines. Osteocyte-derived RANKL, sclerostin, IL-1β, and TNF-α levels are upregulated during skeletal diseases.^159–161 Sclerostin is a key factor in bone resorption. In the periodontitis model, mice deficient in this factor were protected from bone loss. Sclerostin (SOST) enhances the RANKL/OPG ratio and ERK/MAPK in bone.¹⁶²

Additionally, it increases NF-κB activity.¹⁶³ Bacterial lipopolysaccharides (LPS) induce the production of IL-1β, TNF-α, and IL-6, consequently increasing osteocyte-mediated osteoclast formation by upregulating RANKL and JAK2 expression.¹⁶⁴ Serum obtained from patients with rheumatoid arthritis contains a cocktail of pro-inflammatory cytokines. When ex vivo osteocyte cultures were treated with serum from RA patients, these sera induced the expression of SOST, IL-1β, TNF-α, and DKK.¹⁶⁵ Evidently, SOST and DKK1 block bone regeneration.¹⁶⁶ Exogenous TNF-α treatment induces upregulation of IL-1β, TNF-α, IL-8, IL-6, and FGF23 gene expression in human osteocytes.¹⁶⁷ Despite this, the mechanisms underlying the correlation between pro-inflammatory cytokines and osteocytes in the disease context remain poorly understood. However, this information would help design effective therapy for RA patients.

Cytokines Probably Regulate Bone Remodeling by Activating microRNAs

There is a strong association between microRNAs and cytokines in the regulation of bone remodeling. Pro-inflammatory cytokines are expressed at higher levels, accompanied by increased levels of certain microRNAs. MicroRNAs are epigenetic regulators that play a crucial role in regulating gene expression by targeting mRNAs to suppress their translation. Evidence indicates that miRNAs can impact osteogenesis, osteoclastogenesis, and also bone repair. The major signaling pathways regulated by miRNAs include the RANKL-OPG-RANKL, M-CSF, Jagged/Notch, Wnt/β-catenin, and bone morphogenetic protein (BMP) pathways.¹⁶⁸ Apart from that, microRNAs, along with pro-inflammatory or anti-inflammatory cytokines, were observed to aggravate disease symptoms in coronary artery disease (CAD), coronary atherosclerosis (CA), unstable angina (UA), acute myocardial infarction (AMI), and ST-segment-elevated myocardial infarction (STEMI).¹⁶⁹ Therefore, it is possible that, when both cytokines and microRNAs are taken into account, they may exert prominent control over bone remodeling as well.

For example, miR-21 decreases connexin 43 expression, a molecule responsible for bone growth, by interacting with RANKL and high-mobility group box-1. This entire process protected osteoclasts from apoptosis and also enhanced their differentiation.¹⁷⁰ miR-21 was found to increase along with TNF-α, IL-1β, IL-18, IL-8, and CRP in CAD, and IL-6 in AMI. miR-21 was downregulated by estrogen, thereby increasing FasL and caspase-3 protein during osteoclast differentiation. This indicates that miR-21 levels regulate the ERα pathway.¹⁷¹ miR-31 was reported to regulate cytoskeletal organization and bone resorption by targeting RhoA protein.¹⁷² With decreased miR-31, TGF-β levels also decreased in CAD.¹⁶⁹ miR-155 was reported to be upregulated in osteoporosis, and it ultimately targets the leptin receptor. Downregulation of miR-155 suppresses the RANK-OPG-RANKL signaling axis and promotes bone formation.¹⁷³ This downregulation could also occur by targeting purine-rich-binding protein 1 and microphthalmia-associated transcription factor.¹⁷⁴ Interestingly, miR-155 also showed a strong correlation with TGF-β levels.¹⁶⁹ According to recent investigations, miR-133-3p is a positive regulator, whereas miR-155-5p and miR-146-3p are negative regulators of osteoclast differentiation, as they suppress the p38-MAPK and RANKL-induced pathway.¹⁷⁵ miR-155 levels change with the expression levels of pro-inflammatory cytokines, such as IL-1β and TNF-α.¹⁶⁹ While miRNA-34a was targeted by transforming growth factor beta-induced factor 2, this led to increased bone resorption, indicating that miRNA-34 can be a crucial component in suppressing osteoclast activity.¹⁷⁶ In disease models such as CAD and AMI, miRNA-34 is upregulated by elevated levels of pro-inflammatory cytokines.¹⁶⁹

miR-182 is a novel microRNA that enhances osteoclast formation by promoting the RANKL pathway, suggesting that it could be another therapeutic candidate for managing inflammatory responses in skeletal diseases.^177,178 Inhibition of this molecule reduced RANKL-induced bone resorption and osteoclast differentiation in rheumatoid arthritis.¹⁷⁸ In contrast, miR-182 was downregulated, accompanied by decreased TGF-β levels.¹⁶⁹ miR-17/20a cluster inhibits glucocorticoid-induced osteoclastogenesis.¹⁷⁹ miR-17 shares a correlation with TGF-β.¹⁶⁹ Another novel microRNA, miR-106b, was observed to suppress osteoclast differentiation and osteolysis by inhibiting the RANKL pathway.¹⁸⁰ In contrast, in coronary artery disease, pro-inflammatory cytokines increase with elevated miR-106b levels.¹⁶⁹ Another study reports that when 34a-5p was delivered via a recombinant adeno-associated viral vector, it increased mass. This miRNA could be targeted to manage both post-menopausal and senile osteoporosis.¹⁷⁶ However, this microRNA was observed to increase along with IL-1β, TNF-α, IFN-γ, and IL-18.¹⁶⁹

Analysis of synovial tissue from patients with rheumatoid arthritis revealed that TNF-α upregulated miR-221-3p in exosomes.¹⁸¹ Increased miR-221-3p expression decreases osteoblast differentiation in calvaria and osteogenesis by affecting Wnt and BMP signaling pathways.¹⁸¹ If the Wnt pathway is suppressed for some reason, it dysregulates the functions of miR-210 and miR-135,¹⁸² since these microRNAs correlate with pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-17, and also with IL-8, IL-2, IL-9, IL-2, and IL-10.¹⁶⁹ miR-30 and miR-133 were also observed to contribute to the RANK/RANKL pathway by favoring osteoclastogenesis¹⁸³ and enhancing cytokines such as IL-1.¹⁶⁹ At the same time, miR-133 also increases IL-10 expression.¹⁶⁹

miR34 serves as a post-translational regulator of Notch signaling during osteoclast differentiation and also influences osteoblastogenesis.¹⁸⁴ miRNA-34 inhibits osteogenesis by blocking the differentiation of osteoblasts and hMSCs.¹⁸⁵ miR-34a can increase IL-6 and TNF-α.¹⁶⁹ While miR-146a was expressed in chondrocytes, it decreased IL-1β levels and, consequently, reduced inflammation and joint degradation in OA.¹⁸⁶ miR-181b-5p plays a critical role in osteogenic differentiation of MSCs by interacting with the Notch pathway.¹⁸⁷ miR-181b-5p has an association with IL-6. miR-181b-5p decreases with decreased IL-¹⁶⁹ miR-199a suppresses chondrocyte differentiation by targeting SMAD1.¹⁸⁸ However, miR-199a was observed to increase levels of IFN-γ, TNF-α, IL-1β, and IL-18.¹⁶⁹ miRNA profiling of chondrocyte differentiation showed that miRNA-20b, miR-345 and miR-146 target TGF-β/BMP pathway, causing osteoarthritis.^189–191 Therefore, beyond doubt, there are a bunch of miRNAs that share a correlation with pro-and anti-inflammatory cytokines in terms of their level of expression. However, whether they regulate each other’s expression remains to be confirmed. Besides, most of this correlation needs to be evaluated in skeletal diseases, as they offer new therapeutic opportunities.

Probable Role of Epigenetic Modifications in Cytokine-Mediated Bone Remodeling

There are a few pro- and anti-inflammatory cytokines that modulate the function of epigenetic regulators through signaling cascades, thereby enabling cells to respond and exert their effects through epigenetic modifications.¹⁹² These modifications can play a significant role in the development of disease conditions. These phenomena have been observed in the context of tumorigenesis and cancer progression,¹⁹³ which disrupts bone homeostasis as well. For example, IL-6 triggers NF-κB and STAT-3-dependent pathways that enhance histone deacetylase 1 (HDAC1) expression and activity, leading to hypermethylation.^194,195 This signaling pathway ultimately inhibits adhesion and apoptosis, promoting tumor formation and metastasis. It also represses the expression of tumor suppressor genes.¹⁹⁵ Similarly, IL-1β also increases methyltransferase activity, leading to promoter CpG island methylation,¹⁹⁶ especially of the IL-6 and IL-8 pro-inflammatory cytokine genes. Ablation of DNMT3b resulted in a reduction of CpG island methylation at the promoter region of pro-inflammatory genes. In colon cancer, IL-1β was observed to restructure the DNA methylome by increasing DNMT3a expression.¹⁹⁷ TGF-β, being an anti-inflammatory cytokine, activates DNA methyltransferases.¹⁹⁸ Histone methyltransferase EHMT2 collaborates with H3K9me3 to silence certain target genes, such as E-cadherin.^199,200 The association of TGF-β with H3K9me2 can also result in trimethylation of H3K4 and H3K36. TGF-β can trigger epithelial-to-mesenchymal transformation in mammary epithelial cells by increasing SIRT1 expression, leading to histone acetylation and suppression of miR-200a.²⁰¹ These examples collectively strengthen the argument that both pro- and anti-inflammatory cytokines perform the same functions within the epigenetic machinery and thus serve the same functions; however, beyond cancer, these effects are yet to be evaluated in skeletal diseases that involve bone damage and chronic inflammatory responses.

Metabolic Pathways Involved in Cytokine-Mediated Bone Remodeling

A balanced relationship between osteoclasts and osteoblasts maintains the continuous cycle of bone formation and resorption, which contributes to bone remodeling. The cytokine network mediates this balance. These cytokines can induce significant changes in the energy metabolism of bone cells, which constitutes a core area of osteoimmunology.^202,203

Osteoclastogenesis, the differentiation of macrophage/monocyte precursors into multinucleated osteoclasts, is a highly energy-consuming process that involves significant metabolic changes (Table 1). The main cytokines involved in this process are already discussed in detail above; for example, TNF-α, IL-1β, and IL-6 can strongly potentiate osteoclast differentiation by upregulating RANKL expression on stromal cells, or else they increase osteoclast precursors’ sensitivity to RANKL.^3,204 The downstream signaling pathways, such as NF-κB, MAPK, and PI3K/Akt, are also activated during osteoclastogenesis. This cytokine-related activation mediates many metabolic shifts. Similarly, most cytokines have dual effects, which are also involved in osteoblast formation (Table 2). During the formation of a large volume of bone matrix, extensive cell proliferation is required, which is mediated by a shift in cell metabolic states.²⁰⁵ It is yet to be confirmed whether these metabolic pathways are directly affected by cytokine-mediated bone damage. However, the link between cytokines and metabolic pathways could be exploited to improve bone degeneration in inflammatory skeletal diseases.

Table 1 Metabolic Pathways Involved in Osteoclast Differentiation

Table 2 Metabolic Pathways Involved in Osteoblast Differentiation

Role of Cytokines in Infection-Mediated Bone Damage

In the previous sections, we observed that cytokines play a pivotal role in modulating bone cell differentiation and thereby significantly influence bone remodeling. Next, we intend to dive into the implications of cytokine-mediated regulation in bone homeostasis in disease contexts. The role of cytokines in the exacerbation of inflammatory skeletal diseases such as rheumatoid arthritis, osteoarthritis, and osteoporosis is already well-documented and profoundly acknowledged. In contrast, the association between cytokines and infection-mediated bone diseases is still emerging and warrants further investigation into potential therapeutic avenues.

In this section, we discuss the involvement of the cytokine network in aggravating the bone damage in infection-mediated skeletal disorders.

Septic Arthritis (SA)

SA is an acute, joint-destructive disease in which the joint loses crucial components, such as glycosaminoglycans, due to infection.²⁰⁹ The occurrence of SA is 2–10 cases per 100,000 individuals. However, the incidence is 15-fold enhanced in rheumatoid arthritis patients.^210–212 Staphylococcus aureus is the main causative agent for this disease. Once the bacteria enter, they release toxins, virulence factors, adhesins, and superantigens, triggering a local immune response.²¹³ This immune response leads to the elimination of the infection but causes permanent joint damage.

A systemic and local inflammatory response was observed in patients suffering from SA. In a study, IL-6 levels were 28 and 525 times higher in patients’ serum and synovial fluid, respectively, compared with the healthy control group. In SA, the immune system responds to the proteoglycan present in the cell wall of S. aureus, releasing TNF-α, IL-6, and IL-1β. Bacterial DNA also initiates an intense inflammatory response.^214,215 Superantigens derived from bacteria activate T cells, leading to the release of IL-2, IFN-γ, and TNF-α.^216,217

Osteomyelitis

Osteomyelitis is a disease that involves severe bone damage, and this disease is challenging to treat, especially if caused by methicillin-resistant S. aureus strains.²¹⁸ In this disease, the treatment often fails, leading to a chronic therapy-refractory infection. S. aureus invades bone cells and, in later stages, converts itself to a metabolically inactive form, making it challenging to eliminate from the body.²¹⁹ Osteomyelitis causes severe bone damage, inflammation, pain, disabilities, and morbidity. S. aureus infection can be acute or chronic, forming a biofilm in the bone. In both cases, the bacteria differ in their virulence. S. aureus invades osteoblasts and destroys these cells using its cytotoxic properties.²²⁰

The immune system exhibits a profound inflammatory response against S. aureus infection. While S. aureus enters and colonizes bone, resident macrophages release chemotactic factors that attract polymorphonuclear neutrophils (PMNs) to the site.^221,222 PMNs effectively kill bacteria through phagocytosis and oxidative bursts. The release of pro-inflammatory cytokines, such as TNF-α and IL-1β, together with CXCL2 and CXCL3 released by macrophages, recruits PMNs to facilitate the clearance of the pathogen.²²³ Superantigen (SpA) released by bacteria binds to TNF-α receptor 1 (TNFR1) on osteoblasts, leading to increased apoptosis in these cells and, subsequently, decreased bone formation^224,225 (Figure 2). TNF-α, released by macrophages, enhances osteoclast differentiation and bone resorption.²²⁶ These resorption lacunae and necrotic bone fragments are evident in biopsies from patients with human osteomyelitis.²²⁷ SpA binds to TNFR1 on osteoblasts, upregulating the expression of RANKL.^222,225 PMNs also increase RANKL expression by activating toll-like receptor 4 (TLR4).²²⁸ PMN also induces IL-8 release from osteoclasts, thereby favoring osteoclastogenesis and bone resorption. In addition to the infection, the persistent release of IL-6, IL-1β, and TNF-α by immune cells also drives osteoclast differentiation and bone resorption.^222,229 Therefore, continuous inflammation is another contributor to bone damage in osteomyelitis.

Figure 2 Pro-inflammatory cytokines lead to severe bone damage in osteomyelitis. During S. aureus infection, bacteria reach to bone through the blood. (A) S. aureus infects osteoblasts and produce SpA and it binds to TNF-α receptor 1 on osteoblasts, which in turn leads to upregulation of RANKL expression. (B) SpA induced apoptosis of osteoblasts releases S. aureus in the microenvironment, leading to infection of neighboring healthy cells, and this cycle repeats. (C) During infection macrophages release TNF-α to clear pathogen. (D) Chemotactic factors produced by tissue macrophages drives the recruitment of polymorphonuclear cells (PMNs). TNF-α and other proinflammatory cytokines released by macrophages and PMNs enhances osteoclast differentiation and promotes bone resorption.

Periodontitis

Periodontitis is an infectious inflammatory disease characterized by the formation of bacterial biofilm on the teeth, ultimately leading to gingivitis.²³⁰ Porphyromonas gingivalis affects the entire oral microbiota. Other pathogens can also disrupt the oral microbiome and cause periodontitis, such as Tannerella forsythia.^231,232 Once this infection progresses, the space between the gingiva and the tooth, known as the periodontal pocket, begins to increase, providing room for the growth and survival of non-commensal bacteria. Periodontitis can be chronic and aggressive.²³³ Periodontal disease results in severe bone damage, tooth loss, and a significant reduction in jawbone volume.

The pro-inflammatory cytokine environment is a major contributor to jawbone loss in periodontitis. An increased presence of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, and IL-12, and enhanced levels of IL-4 and IL-10 were observed.²³⁴ The oral mucosa is lined by an epithelial layer that participates in immune defense by expressing pattern recognition receptors (PRRs) that recognize pathogens via pathogen-associated molecular patterns (PAMPs).^235,236 Apart from epithelial cells, fibroblasts play a significant role in the immune response against pathogens. Fibroblasts release matrix metalloproteinases and proinflammatory mediators like IL-8, IL-6, and prostaglandin E2^237,238 (Figure 3). IL-1 and IL-8 act as chemoattractants for immune cells, such as neutrophils, which phagocytose bacteria and release reactive oxygen species (ROS) to destroy the pathogen. In adaptive immunity, Th1 cells release TNF-α, IFN-γ, IL-2, IL-12, and IL-1, while Th2 cells express IL-4, IL-5, and IL-13 to induce a humoral immune response.²³⁹ Then, Th17 cells produce IL-17, which initiates the inflammatory response and recruits neutrophils.²⁴⁰ Therefore, immune cells and cytokines play a pivotal role in the pathogenesis of periodontitis.

Figure 3 Increased inflammation in periodontal cavity leads to significant bone damage in periodontitis. Bacteria migrate from blood circulation into the periodontal cavity and colonized. (A) Fibroblasts release matrix metalloproteinase 3 (MMP3), IL-6, IL-8, and prostaglandin E2 (PGE2) that mediate inflammation and recruits PMNs and macrophages and promotes induction of ROS production from neutrophils. (B) Activated macrophages can also contributes to the activation and differentiation of T cells. (C) T cells differentiated in the tissue microenvironment releases inflammatory cytokines. These cytokines induce inflammation and recruit neutrophils. The entire process ultimately leads to increased inflammation, enhanced osteoclast differentiation, and increased bone resorption.

Chikungunya

Chikungunya fever is caused by Chikungunya virus (CHIKV), an arbovirus. The most visible symptoms are a high fever, headache, vomiting, skin rashes, and severe muscle and joint pain. The symptoms subside within a few days or weeks if the infection is acute; however, a chronic infection may persist for months.²⁴¹ It is reported that more than 40% of chikungunya patients develop arthritic symptoms, including bone loss and joint pain.^242,243 IL-6, IL-1, and MCP-1 are produced during the inflammatory process in chikungunya, and these cytokines promote osteoclast differentiation and function. The functionality of the osteoblast is damaged in parallel.

T lymphocytes actively participate in inducing the cellular infiltration and inflammatory process. In Chikungunya, CD4⁺ T lymphocytes release a wide range of pro-inflammatory cytokines and chemokines, including IL-1, IL-6, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, IP-10, and MIP-1²⁴⁴ (Figure 4). These cytokines may contribute to inflammation, pain, and bone damage. The T-cell response was detected in patients with chronic CHIKV infection.²⁴⁵ However, this effect is more profound in older individuals.²⁴⁶ Monocytes and macrophages are the major cell types that infiltrate the site of inflammation. Macrophages are active reservoirs for the virus and produce cytokines such as IL-6, TNF-α, and prostaglandins.^247–249 Monocytes are precursors for osteoclast differentiation. Following CHIKV infection, monocyte chemoattractant protein-1 (MCP-1) levels are elevated, directly contributing to enhanced osteoclast differentiation and bone resorption.²⁵⁰ While the Chikungunya virus infects osteoblasts, it induces the production of IL-6, which, in turn, alters the RANKL/OPG ratio. This phenomenon favors the differentiation of monocytes into osteoclasts, which initiate bone resorption.^251,252

Figure 4 Chikungunya infection causes severe bone damage through the release of pro-inflammatory cytokines. (A) Upon viral entry, T cells release various cytokines, including IL-1, IL-6, IL-10, IL-12, IL-15, IL-17, IL-18, TNF-α, and MIP-1. (B and C) monocytes and macrophages release IL-6, TNF-α, and prostaglandin. These factors mediate inflammation. Pro-inflammatory cytokines released by T cells induce macrophages to release more RANKL. T cells also begin to express higher levels of RANKL. (D) RANKL expression exceeds OPG expression. (E) Thus, RANKL, produced by T cells, monocytes, and mesenchymal stem cells (MSCs), promotes osteoclast differentiation and bone resorption. Note: The green arrow indicates upregulation, and the red arrow indicates downregulation.

HIV Infection

Currently, there are more than 36 million people around the world who are suffering from human immunodeficiency virus (HIV) infection, and many of them can access antiretroviral therapy (ART) now.²⁵³ ART reduces viral load but, as a side effect, can affect the patient’s bone mineral density (BMD). Long-term battles with HIV infection and ART both contribute to several skeletal abnormalities, such as osteopenia, osteomalacia, osteoporosis, fractures, and other bone disorders.²⁵³ MSCs express receptors for CD4⁺ T cells and coreceptors CCR5 and CXCR4, which are susceptible to HIV infection.²⁵⁴ A high viral load makes MSCs pro-adipogenic, favoring adipogenesis over osteogenesis. The HIV transactivator (Tat) protein is involved in this process. The function of this protein is to regulate the reverse transcription of the viral genome. This protein enhances the activity of RANKL and M-CSF, thus increasing osteoclast differentiation.

Additionally, osteoclast-specific genes, such as CTSK, TRAP, and calcitonin receptor, exhibit increased mRNA expression.^255–258 Tat and HIV-negative factor (Nef) decrease the number of MSCs differentiating into osteoblasts as cells undergo senescence, accompanied by oxidative stress and mitochondrial dysfunction.²⁵⁹ In HIV, lentiviral protein R (Vpr) contributes to the nuclear import of the pre-integration complex. This protein increases RANKL expression in peripheral mononuclear cells, thereby increasing osteoclast activity. Exogenous and endogenous glucocorticoids synergize with RANKL to promote osteoclast differentiation.²⁶⁰ The production of ROS and TNF-α promotes osteoclast formation and bone resorption.^261,262 Raynaud-Messina et al showed that HIV can infect osteoclast precursors at different stages of differentiation as they get transferred from infected T cells. The infected precursors act as viral reservoirs and exhibit enhanced migratory ability. Post-infection, the podosomes get enlarged, and the osteolytic potential gets enhanced in the “sealing zone” of the osteoclasts. Consequently, the bone-resorbing capacity of osteoclasts increases manifold.²⁶³ Several other viral proteins are involved in disrupting homeostasis during bone remodeling. p55-gag and gp120 decrease calcium deposition, ALP activity, and the secretion of BMP-2, 7, along with an increase in RANKL²⁶⁴ (Figure 5). HIV infection establishes a positive feedback loop to increase RANKL, which ultimately leads to an osteopenic condition.²⁶⁵

Figure 5 HIV infection induces bone damage. (A) Upon entry, HIV first infects CD4+ T cells, where RANKL expression increases. (B) MSCs express receptors for CD4+ T cells and coreceptors CXCR4 and CCR5. (C) In infected CD4+ T cells, HIV encodes Tat, Nef, Vpr, p55-gag, and gp120, all of which enhance adipogenesis. (D) and decrease osteogenesis. The expression of osteoblast markers, such as alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protein-7 (BMP-7) decreases, while RANKL expression increases. (E–G) HIV, T cells, and osteoblast-derived RANKL increase osteoclast differentiation. Osteoclast-specific genes, such as TRAP, cathepsin K, and calcitonin receptors, are upregulated. (H) B cells release more RANKL in later stages, contributing to osteoclast differentiation. (I) Th1 cells and macrophages produce pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-α, which enhance osteoclast differentiation. All cell types increase bone resorption. Note: The green arrow indicates upregulation, and the red arrow indicates downregulation.

The gp120 protein induces apoptosis in osteoblasts by upregulating TNF-α.²⁶⁶ The gp120 enhances expression of the Wnt pathway antagonist Dickkopf-1 (Dkk1) and degrades intracellular β-catenin, thereby suppressing alkaline phosphatase activity and cell proliferation.²⁶⁷ In HIV infection, B and T lymphocytes undergo dysfunction. RANKL expression by B cells is upregulated, leading to an abrupt shift in the OPG/RANKL ratio. Similarly, OPG production from CD4⁺ T cells is downregulated.²⁶⁸ Enhanced RANKL production leads to the development of osteopenic conditions in patients.²⁶⁹ Infection with HIV also disturbs the cytokine production. The production of pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-α, is increased during this infection. This abnormal cytokine generation is likely to contribute to increased osteoclast formation, ultimately leading to bone loss and osteopenia.²⁷⁰

Therapeutic Targeting of Cytokines in Alleviating Infection-Mediated Skeletal Disorders

Pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, and TNF-α, play a crucial role in exacerbating inflammation and bone damage caused by certain infections. Pro-inflammatory cytokines, along with RANKL, promote osteoclast differentiation and contribute to skeletal damage. Therefore, a combination therapy of antibiotics and agents targeting TNF is beneficial for managing bone loss caused by pathogenic infections, such as those caused by Staphylococcus bacteria. IL-1 and IL-6R antagonists were also reported to be helpful.²⁷¹ RANKL is the primary factor for osteoclastogenesis and bone resorption. Therefore, inhibiting RANKL would be beneficial in preventing bone resorption. Denosumab is a monoclonal IgG2 antibody that blocks RANKL/RANKL signaling.²⁷² Denosumab inhibits bone destruction by suppressing bone-related factors and cytokines. It has been successfully used in the treatment of inflammatory joint disorders,²⁷³ rheumatoid arthritis,²⁷⁴ and in the prevention of fractures in postmenopausal women with osteoporosis.²⁷⁵ Denosumab rescues trabecular and cortical bone. However, because RANKL is expressed on T and B cells, it suppresses their activity, thereby increasing the likelihood of infection.²⁷⁶ Denosumab-binding peptide fused with the diphtheria toxin T domain (DTT-RANKL(220-245)3, also known as the DR3 vaccine) has shown a positive response. It may be explored for the prevention and treatment of osteoporosis or other bone-resorptive diseases.²⁷⁷ Blocking RANKL with OPG administration showed some positive effects in the septic arthritis model. OPG and cloxacillin significantly decreased osteoclast activity in infection-induced osteoporosis.²⁷⁸ Therefore, OPG could be combined with antibiotics as a promising therapeutic option.

Anti-cytokine therapies were also adopted for periodontal disease. Different approaches can achieve cytokine downregulation. (1) Administration of soluble receptors, which will prevent the signaling cascade. (2) Receptor antagonists bind to the cytokine receptors, blocking the receptor-ligand interactions. (3) Inhibitors bind to the cytokines to prevent their binding to the respective receptors. Bortezomib, infliximab, etanercept, and denosumab are used to combat inflammation in periodontitis.²⁷⁹ Tocilizumab is a monoclonal antibody that binds to the human interleukin-6 (IL-6) receptor, thereby blocking IL-6’s pro-inflammatory activity. Tocilizumab reportedly prevents alveolar bone loss in periodontitis.²⁸⁰ The injection of human recombinant IL-11 slowed the progression of bone loss in dogs with periodontal disease.²⁸¹ Psoralen is a drug that suppresses the transcription and secretion of IL-1β and IL-8 in experimental animal models of periodontitis.²⁸² However, there are certain limitations to the anti-cytokine therapies. The trials have not yet been performed on a large group of patients. The anti-cytokine drugs have been tried in autoimmune diseases only. Research on anti-cytokine therapy is currently limited to in vitro and animal models. Several anti-TNF-α antibodies, including infliximab, certolizumab, adalimumab, and golimumab, have been successfully used in RA patients and have shown a positive impact on bone metabolism, thereby preventing further bone loss.²⁸³ The pro-inflammatory cytokine IL-9 has also been recently shown to have osteoclastogenic function, making it an interesting target for controlling bone loss-related disorders.²⁸⁴

Conclusions

Cytokines mediate communication among cells within the same organ or between different organs. This mode of communication is crucial in maintaining the overall homeostasis. Besides, cytokine production is an indispensable factor in controlling disease progression, as it orchestrates the immune response to eliminate the pathogen and/or initiate healing. However, excessive cytokine activity can have detrimental consequences. How the cytokine network regulates bone formation and resorption is undoubtedly an exciting field to explore. Emerging evidence strongly suggests that pro-inflammatory cytokines play a key role in inducing bone damage by activating the NF-κB, MAPK, and PI3K/AKT pathways, which recruit the transcription factor NFATc1. This is how pro-inflammatory cytokines enhance osteoclast differentiation and promote bone degradation.

On the other hand, anti-inflammatory cytokines preserve bone mass by suppressing osteoclastogenesis and bone resorption. Despite extensive research to date, a comprehensive, systematic approach is still required to understand cytokine biology and its impact on bone homeostasis. Intriguingly, cytokines, both individually and in combination, can serve as diagnostic markers for various bone diseases, but this requires further study and monitoring across different disease stages. In addition to activating different signaling pathways, cytokines also affect epigenetic modifications, regulate microRNA activity, and influence various cellular metabolic pathways; these downstream effects can be leveraged for therapeutic benefit. However, they must be examined in the context of skeletal disease.

Anti-cytokine therapy is a recent choice to manage the extensive bone damage caused by a constant inflammatory environment associated with skeletal diseases. Several monoclonal antibodies, such as denosumab, infliximab, and tocilizumab, have been launched and are being tested for diseases such as osteoporosis and RA. These antibodies successfully block the activities of RANKL, TNF-α, and IL-6; however, their efficacy is still under investigation. Pro-inflammatory cytokines also play a critical role in promoting inflammation and bone loss in infectious diseases. Reportedly, bone damage can occur due to both bacterial and viral infections, driven by the persistent inflammatory environment post-infection. Therefore, anti-cytokine therapy shows promise in managing inflammation and infection-mediated bone degeneration, but further research is necessary before reaching any definitive conclusions. Anti-cytokine treatment has its share of advantages and disadvantages.

Furthermore, an individual’s gender, age, and neuropsychological status significantly impact cytokine levels. The mechanisms by which cytokines are produced during infection, autoimmunity, and alloimmunity remain to be fully explored. Interestingly, anti-cytokine therapy was observed to affect osteocytes’ mechanosensing. Therefore, intra-organ crosstalk must also be considered when testing anti-cytokine therapies in skeletal diseases.