5. Neutrophils as a Target for Osteoarthritis Treatment
As one of the major immune cells, neutrophils have a dual role in the pathogenesis of arthritis. On the one hand, these cells act as a mediator with considerable potential to destroy the articular cartilage and other joint components by releasing pro-inflammatory cytokines, destructive enzymes, and chemotactic factors that stimulate the migration of other immune cells. On the other hand, neutrophils have long-term homeostatic functions, including orchestrating the resolution of inflammation and contributing to articular cartilage repair as a regulator of immune response and a player in changing their phenotypic plasticity [
24,
91,
92]. Because of the intimate role of neutrophils in arthritis, they have emerged as therapeutic targets [
2]. Several aspects of neutrophil biology may be therapeutically targeted, including production, recruitment, function, and apoptosis (
Supplementary Table S2) [
93,
94].
Neutrophils contribute to producing and releasing many cytokines, chemokines, and enzymes within the joint that promote OA progression [
2]. Non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and hyaluronic acid are the most commonly used medications for OA to alleviate joint pain, inflammation, and function [
95,
96]. NSAID-mediated anti-inflammatory mechanisms independent of inhibiting COX and PGE2 release have been proposed [
97]. These medicines inhibit neutrophil aggregation and degranulation [
98]. Moreover, NSAIDs can reduce C5a- and CXCL8-induced neutrophil migration and F-actin polymerization through integrin downregulation or PI3K/Akt pathway [
97]. Despite the immunosuppressive effect glucocorticoids have on immune cells, they exert multiple and even contradictory effects on neutrophils. They can inhibit or induce apoptosis in neutrophils. Likewise, glucocorticoids may have anti-inflammatory or pro-inflammatory effects on neutrophils [
99]. Glucocorticoids also influence neutrophils’ maturation, extravasation, adhesion, metabolism, and activation via signaling pathways. Hyaluronic acid exerts protective effects against arthritis through different mechanisms [
99,
100]. Hyaluronic acid interacts with joint cells, including synoviocytes, chondrocytes, osteocytes, and immune cells, and affects inflammatory mediators [
101,
102]. AKT has a pivotal role in OA, and it was found that hyaluronic acid significantly reduced the p-AKT expression level in synovial-fluid neutrophils. Furthermore, hyaluronic acid can reduce the levels of phosphorylated p38MAPK, NF-κB, p53, Bax, and Caspase-3 in synovial fluid neutrophils that indicates the modulatory effect of hyaluronic acid on pro-inflammatory responses and pro-apoptotic events [
103].
Different anti-cytokine strategies, including antibodies against pro-inflammatory cytokines, such as TNF-a and IL-1β, or the use of anti-inflammatory cytokines, namely TNF-β, IL-4, and IL-10, have been investigated for arthritis treatment. TNF-α is a pro-inflammatory cytokine produced by neutrophils with a considerable role in inflammatory arthritis and cartilage and bone degeneration [
67,
104,
105,
106,
107]. Different TNF-α blockers have been evaluated for treating arthritis, especially inflammatory RA [
105,
106]. A reduction in peripheral blood neutrophil count was reported following treatment with anti-TNF-α in arthritic patients [
107]. TNF-α inhibitors may affect neutrophils in different ways. They attenuate the generation of pro-inflammatory cytokines, chemokines, and MPO by neutrophils and also contribute to the upregulation of adhesion molecules expression and priming of respiratory burst in adherent neutrophils [
108,
109].
Moreover, anti-TNF-α agents can reduce neutrophil ROS production and the influx of neutrophils from inflamed joints [
108]. CD69 is a type II membrane protein expressed on T and B cells, platelets, eosinophils, and activated neutrophils with a crucial role in inflammatory joint diseases [
109,
110]. Anti-TNF-α has been capable of inhibiting CD69 expression on arthritic neutrophils and downregulating neutrophil chemoattractant IL-8. Therefore, these cells can be targets for anti-TNF-α treatment [
111,
112]. It was demonstrated that TNF-α inhibition did not modify neutrophil immune functions against pathogen agents and was tolerated in terms of serious adverse effects. However, it has been suggested that neutropenia caused by TNF inhibitors may be due to accelerated apoptosis, and patients at higher risk of developing neutropenia had a low baseline neutrophil [
107,
111,
113].
IL-1 family, particularly IL-1β and interleukin-1 receptor, is the key component linked to the pathogenesis of arthritis [
114,
115]. Consequently, the reduced synthesis of proteoglycans by chondrocytes and increased synthesis of proteolytic enzymes, NO, and other cytokines mediated by IL-1β cause cartilage destruction in OA [
116,
117]. This cytokine induces neutrophil production, recruitment, degranulation, and NETosis. Moreover, it delays neutrophil apoptosis through different mechanisms, such as upregulating neutrophil chemoattractants (i.e., CXC- and CCL- chemokines), inducing adhesion molecule expression and local chemokine production (IL-8), as well as upregulating anti-apoptotic agents (i.e., Bcl-2 family and Mcl-1) [
37,
118]. Furthermore, releasing IL-1β by neutrophils causes positive feedback during the autoinflammatory process by binding to the type 1 IL-1 receptor [
119]. IL-1Ra inhibits cytokine-induced catabolism, and IL-1 deficiency protects joints from inflammation in induced arthritis [
116,
120]. It has been proposed that the therapeutic function of IL-1 receptor antagonist (IL-1Ra) may be more effective by reducing neutrophil recruitment into the joint cavity rather than increasing apoptosis or inhibiting the activation of neutrophils.[
118]. Evidence of the beneficial role of IL-1Ra as a disease-modifying OA drug (DMOAD) was demonstrated in experimental autoimmune arthritis and OA models [
120]. Anakinra is a recombinant form of IL-1Ra indicated for improving clinical signs and slowing the progression of structural damage in OA, RA, and other immune-mediated arthritis types [
113,
116,
121]. A recent in vitro study suggested that anakinra inhibited NET formation in neutrophils, and this process was dependent on IL-1β signaling pathways [
122]. Canakinumab is a human monoclonal anti-IL-1β antibody with an indication for RA and juvenile idiopathic arthritis [
113]. Different studies demonstrated that canakinumab decreased OA symptoms and had a chondroprotective effect. This agent induces neutrophil apoptosis through MAPK14, NF-κB downregulation, and GRP78 upregulation [
123,
124,
125]. Data from an experimental model of arthritis showed that the administration of recombinant IL-37 suppressed joint inflammation associated with the decreased neutrophil influx into the joint parallel with a reduction in neutrophil chemo-attractant chemokines (C-X-C motif) ligand 1 (CXCL1), macrophage inflammatory protein 1-alpha (MIP1α/CCL3), and IL-1α. In addition, reduction in pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and the neutrophil enzyme MPO were related to the therapeutic potential of IL-37 [
121]. Blockade of anti-IL-6 receptors through reducing neutrophil infiltration and NET formation can have a therapeutic role in arthritis [
126].
Recent evidence identified that miRNAs play an important role in regulating cartilage and bone homeostasis, catabolism, and anabolism ad repair [
127,
128]. The miRNAs are a group of endogenous small noncoding RNAs, and their up- or downregulation has been suggested to be linked to the pathogenesis of arthritis. Anti-cytokine therapy can affect miRNAs due to interactions between cytokines and them [
129,
130,
131]. It has been suggested that treatment with anti-TNF-α and anti-IL-6 receptors modulates miRNA levels in neutrophils and attenuates inflammation [
132]. In vitro treatment of RA neutrophils with anti-TNF-α and anti-IL-6 receptors has been accompanied by diminished miRNA levels in neutrophils and enhanced inflammatory profile [
133].
Although cytokine-modulating therapies have been considered for arthritis treatment, a question of interest is whether cytokine-modulating therapies with just one cytokine or chemokine will be sufficient to stop inflammation and improve OA. It is important to note that neutrophils and other immune cells contribute to releasing many cytokines and chemokines with a potential synergistic effect in a cascade reaction during OA initiation and progression. Therefore, blocking one member can interrupt this synergy [
3,
134], and different pro-inflammatory and anti-inflammatory cytokines and their interactions can be considered for new therapeutic approaches to arthritis [
135].
NE, secreted by neutrophils during inflammation, can potentially be targeted for OA treatment. NE is a degenerative protease that can induce cartilage destruction and pain correlated with arthritis severity during OA [
43,
136]. NE participates in neutrophil migration by cleaving adhesion molecules and modifying chemokine and cytokine activity and interaction with specific cell surface receptors [
137]. Sivelestat sodium hydrate is a synthetic, potent, selective inhibitor of NE, which is used for treating acute lung injury and has raised considerable interest in arthritis treatment. Sivelestat can reduce inflammation and pain by inhibiting TNF-α, IL-6, NO secretion and PAR2, p44/42 MAPK activity [
43,
136,
138,
139]. Furthermore, in the OA condition, sivelestat inhibited NF-κB and HMGB1, which is known to be contributed to the NET formation and neutrophil recruitment [
138,
140,
141]. Besides inhibiting NE, targeting the MMPs produced by neutrophils may also be influential for arthritis therapy [
142]. However, the inhibition of MMPs has so far provided only limited therapeutic benefits probably due to the bilateral function of different MMPs in pathological conditions [
93,
143,
144]. Another promising therapeutic approach for arthritis is considering pathways with the ability for NET formation inhibition or have an effect on the component in NETs. [
145]. It was found that polydatin a natural precursor of resveratrol treatment markedly inhibited NET formation mediated by neutrophils and protected the joint against arthritis [
146].
Other studies exist on developing therapies that target the key activators of neutrophils, such as intracellular signaling molecules (e.g., Janus kinase, spleen tyrosine kinase, and p38 MAP kinase) and adenosine receptors [
109]. APPA (apocynin and paeonol) is a plant-derived compound with anti-inflammatory and chondroprotective properties that are considered a novel medication for OA treatment. It is currently being tested in clinical trials for human application [
147,
148,
149]. An in vitro study on the effect of APPA on neutrophils showed that while APPA does not significantly impair neutrophil defense function, it modulates pathological aspects of neutrophil functions. APPA reduces neutrophil degranulation and NET formation. In addition, it dysregulates TNF-α, TNFα-mediated IL-8 expression of and ROS generation by neutrophils, as well as the inhibition of cytokine-driven signaling pathways (i.e., TNF-α-mediated activation of NF-κB and GM-CSF activation of Erk1/2) [
148].
As a hemopoietic growth factor, G-CSF and its receptor are essential in neutrophil release, activation, and function [
150,
151]. It has been shown that G-CSF receptor blockade attenuated the progression of the inflammatory joint disease by blocking neutrophil trafficking with the suppression of chemokines (KC, MCP-1) and pro-inflammatory cytokines (IL-1β, IL-6) production as well as changes to cell adhesion receptors, including decreased CXCR2 and increased CD62L expression. This therapeutic strategy could be achieved without adverse effects on the immune response [
151]. Inhibition of the pro-inflammatory pathway of the CXCR2 receptor via reducing neutrophils to the inflamed joints may be effective in arthritis treatment. Similar successful results have been reported using CXCR1/2 inhibitors on neutrophil recruitment in different inflammatory arthritic models [
152].
Applying mesenchymal stem cells (MSC) for OA treatment holds immense potential and has been increasingly applied as a therapeutic method. MSCs isolated from distinct sources have strongly suggested that MSCs benefit arthritis treatment [
149,
153,
154,
155]. Although stem cells have been proposed to have unique abilities associated with tissue regeneration, the underlying mechanisms of MSCs remain largely unknown. However, other studies have concentrated on the interaction of MSCs with the immune system. [
149,
156,
157,
158]. It seems that MSCs might exert their positive effect on arthritis by modulating neutrophil migration and function. Several mechanisms, including the suppression of NO secretion, decreasing N-Formyl-L-Methionine-L-leucyl-L-phenylalanine, and induction of respiratory burst have been postulated to explain the effect of MSCs on neutrophils [
159]. During the initial stage of inflammation, the first-respondent cells recruited to the inflammation site are neutrophils; their survival is the central arm for inflammation resolution and tissue repair [
159,
160]. It has been reported that MSCs can inhibit neutrophils apoptosis through IL-6, which is signaled by activating STAT-3 transcription factor, downregulation of Bax, and upregulation of MCL-1 [
156,
160]. Secretion of IL-6, IFN-β, and GM-CSF by MSCs can sustain neutrophils viability and function. MSCs also can reduce the adhesion, infiltration, and recruitment of neutrophils through TNF-stimulated gene 6, CXCL2, and CXCR2 [
156,
159,
161]. The crosstalk between MSCs and neutrophils contributes to tissue repair and regeneration [
162,
163]. MSCs can polarize the pro-inflammatory N1 subset into the immune modulatory N2 subset by modulating the extracellular signal-regulated kinase pathway. Furthermore, an interplay between MSCs and neutrophils plays a role in vascular regeneration during the healing process via the effect on IL-6, PDGF, angiopoietin-1, HGF, and VEGF expression [
163]. Chondrogenic progenitor cells (CPCs) are defined as stem cell-like cells in articular cartilage and are identified in different stages of OA. These cells have recently raised great interest in OA treatment because of their self-renewal, multilineage differentiation, and immunomodulatory and phagocytic properties [
164,
165]. Both pro- and anti-inflammatory actions of CPCs have been identified, and different treatment strategies related to CPCs are focused on their effects on the regulation of neutrophils [
2,
156]. Several inflammatory cytokines, chemokines, and MMPs, such as IL-6, CXCL-12, macrophage inhibitory factor, and MMP-13, are expressed by CPCs. CPCs may have an essential role in attracting neutrophils and their degranulation via IL-8 production in the early stage of cartilage injury [
166]. TNF-α and IL-1β released by neutrophils inhibit the migration of CPCs. In contrast, CPCs produce an IL-1 receptor antagonist (IL-1Ra) that inhibits the binding of IL-1β [
2].
Cell membrane-coated nanoparticles (NPs) are recently considered another promising strategy for arthritis treatment [
167]. NPs consist of an NP core coated with membrane derived from natural cells, including white blood cells, and have an excellent biological interface and natural characteristics of source cells properties [
168,
169]. Neutrophil-NPs can absorb and interact with various inflammatory cytokines, including IL-1β and TNF-α. As a result, pro-inflammatory factors are neutralized, and synovial inflammation is inhibited [
170,
171,
172]. Moreover, neutrophil-NPs mimic the natural adhesion between neutrophils and chondrocytes, which increases their cartilage penetration for targeting chondrocytes [
172,
173]. Anti-inflammatory activity and apoptosis inhibition of inflamed chondrocytes have been demonstrated for NPs in OA [
174]. Evaluating the effect of neutrophil-NPs on the arthritis model showed their effectiveness in ameliorating joint damage and suppressing overall arthritis severity [
167]. Neutrophil-based drug delivery systems were applied in treating various inflammatory conditions, including arthritis. This method has considerable biological potential to target different tissue mechanisms, overcome multiple physiological barriers, and enhance accumulation in the target tissue [
175,
176,
177].
Several studies have identified evidence of cellular communication among different types of cells in the joints and their roles in OA pathogenesis [
164,
178,
179]. Extracellular vesicles (EVs) are a novel form of intercellular communication, contributing to the spectrum of physiological and pathophysiological processes by transferring bioactive cargo, including lipids, proteins, nucleic acids, and metabolites, to target cells [
180,
181,
182]. EVs are spherical lipid structures secreted by nearly all cell types. They are categorized into three main subtypes, microvesicles (MVs), exosomes, and apoptotic bodies, for their therapeutic potential [
180,
181,
182,
183,
184,
185,
186]. Nanoenzyme-engineered neutrophil-derived exosomes with the potential to inhibit pro-inflammatory factors production have been shown to alleviate inflammatory stress in fibroblast-like synoviocytes and attenuate joint injury.
Moreover, these exosomes have relieved inflammation synovitis and ameliorated cartilage damage via inducing Th17/Treg cell balance regulation to suppress overall arthritis severity [
187]. Neutrophil-derived microvesicles (NDMVs) have been demonstrated to inhibit inflammation by limiting recipient cells’ immune responses, which could encourage further research into targeting arthritis with NDMVs [
186,
188]. Downregulation of TNFα-induced expression of IL-5, IL-6, IL-8, MCP-1, IFNγ, and MIP-1β using NDMVs internalized by FLS isolated from OA patients, has been reported [
189]. Evidence from an in vitro investigation and an in vivo study suggests that neutrophil MVs enriched in AnxA1 caused TGF-β production, matrix deposition, and chondrocyte homeostasis by inducing FPR2/ALX signaling and protecting the joint. Furthermore, neutrophil EVs reduce the loss of sulfated glycosaminoglycans. They could exert their protective effect on joints via EV mediator Annexin A1 (AnxA1) and its receptor Fpr2/3 on the chondrocyte as well as polarizing macrophages towards a more anti-inflammatory phenotype characterized by higher levels of CD206 and lower expression of MHCII and CD86. Similarly, neutrophil MVs enriched in AnxA1 interact with their receptor formyl peptide receptor 2/ALX and induce TGF-b1 production which causes ECM deposition and chondrocytes protection [
92].