Osteoarthritis (OA) is a progressive, degenerative, condition that affects over 550 million people worldwide–a 113% increase since 1990. The prevalence has doubled in the US over the last 10 years with an economic impact of $136 billion annually (Yelin et al., 2016; Cui et al., 2020). Despite this high prevalence, there remains a lack of effective treatment options that improve quality of life without risk of adverse effects, with current therapies including non-steroidal anti-inflammatories, intra-articular injections, or arthroscopic debridement and cartilage resurfacing techniques. Cellular therapies to treat OA have emerged as an option in both human and veterinary orthopedics with mixed results reported in terms of efficacy (Mezey and Nemeth, 2015; Cortes-Araya et al., 2018; Ghannam et al., 2018; Kwon et al., 2022; Chahla et al., 2016; Harrell et al., 2019; Iijima et al., 2018; Li et al., 2018; Piuzzi et al., 2018; Şahin et al., 2018; Shah et al., 2018; Southworth et al., 2019; Xing et al., 2018). A recent meta-analysis of human randomized MSC trials concluded that overall MSC treatment reduced joint inflammation, improved pain scores, and preserved cartilage integrity, suggesting that MSC cellular therapy is beneficial. Nonetheless, challenges remain, including the use of MSC with greater potency for their intended task, greater uniformity of MSC sources and propagation, as well as improved credentialing of MSC functional properties that correlate with better outcomes (Copp et al., 2023). Heterogeneity within stromal cell populations has been proposed to be partially responsible for the observed variability in therapeutic responses, particularly in the context of variably inflamed recipient environments such as that seen in OA (Cassano et al., 2018). Pre-activation, or “inflammatory licensing,” of mesenchymal stromal cells (MSCs) through priming with their respective ligands has been proposed as a means to generate a homogenous population of immunomodulatory MSCs - thereby potentially improving the consistency of MSC therapy and response to treatment (Szabó et al., 2015; Krampera, 2011; Polchert et al., 2008; Waterman et al., 2012). With the lifetime likelihood to develop symptomatic knee OA currently at 45% and increasing, the need to develop improved strategies towards disease-modification is critical (Murphy et al., 2008).

Expression of pattern recognition receptors (PRRs), specialized proteins that detect exogenous pathogens and endogenous ligands, represent a key link between the immune system and sensory nervous system in response to inflammation or injury, such as that described in OA (Donnelly et al., 2020). The PRR family includes Toll-like receptors (TLR) (e.g., nucleotide oligomerization domain-like receptors, C-type lectin receptors, RIG-I-like receptors, and retinoic acid-inducible gene I receptors) and cytosolic DNA sensors (e.g., STimulator of Interferon Genes or STING). The STING receptor, also known as cGAS-STING, as Cyclic GMP-AMP synthase (cGAS), is the primary enzyme to induce STING pathway signaling. In response to tissue injury, PRRs on immune cells are activated to initiate their respective downstream inflammatory response. Activation of both STING and TLR3 results in production of type I interferons (IFN-Is) (IFN-α, IFN-β, and IFN-κ) in immune cells and sensory neurons following tissue injury or infection (Donnelly et al., 2021; Sun et al., 2022; Wang et al., 2021; Ishikawa and Glen, 2008; Liu et al., 2016), which has been demonstrated to have the potential to be both inflammatory or antinociceptive depending on the disease process (Donnelly et al., 2020; Liu et al., 2016). In the context of specifically enhancing therapeutic efficacy of immunomodulatory MSC therapy, our previous studies have demonstrated that stimulation of MSC with TLR ligands enhanced their immunomodulatory properties to a greater extent than other agonists evaluated, including cytokine secretion of MCP-1 and IL-8 in vitro resulting in resolution of inflammation and reduced bioburden associated with musculoskeletal infection in animal models (Pezzanite et al., 2021; Johnson et al., 2017; Pezzanite et al., 2022). We have more recently expanded this work to evaluate STING pathway agonism in MSC based on the concept that TLR3 and STING pathway agonists may trigger similar immune response pathways, particularly those involving interferon responses. However, to the authors’ knowledge, comparison of these two PRRs to activate and enhance MSC therapy for treatment of OA has not been previously reported.

Therefore, we built upon our previous work with TLR3 activated MSC to evaluate and compare the functional effects of activation of MSCs via TLR3 pathway and the cGAS-STING pathway in the context of intra-articular treatment of OA. The overall objective of this work was to determine whether priming of MSC with agonists of the cytoplasmic (endosomal) TLR3 pathway or the cytoplasmic cGAS-STING pathway exerted similar effects to improve the efficacy of MSC intra-articular therapy, as evaluated in a rodent destabilization of the medial meniscus (DMM) model of OA. Our analysis revealed significant improvements in post-operative voluntary movement parameters and histological outcomes with STING pathway activated MSC and improved histological outcomes with TLR3 pathway activated MSC. RNA sequencing of joint tissues indicated that IFN related pathways were upregulated in the joints of mice treated with either TLR3-activated MSC or STING-pathway activated MSC, with substantially more interferon pathways being upregulated in the STING-pathway MSC treated animals.

Overall, this work provides key new insights into the transcriptomic, structural, and synovial responses to intra-articular treatment with innate immune pathway activated MSC, suggesting that activation of TLR3 pathways or STING pathways in MSC prior to injection can activate multiple joint protective pathways, some of which may involve interferon gene pathway upregulation. Thus, stimulation of IFN pathways in MSC may be a uniquely effective method of licensing MSC for orthopedic applications.

This study was approved by the Institutional Animal Care and Use Committee at Colorado State University (IACUC protocol #3134) and conducted according to the national guidelines under which the institution operates and the NIH Guidelines for the Care and Use of Laboratory Animals (8th edition).

Intra-articular mesenchymal stromal cell products have demonstrated therapeutic potential in preclinical models of OA previously with varied results reported for efficacy. The findings reported here add to the body of literature regarding the use of regenerative therapies to treat OA, highlighting the potential for STING agonism of MSCs to enhance both functional outcomes and structural joint integrity while promoting transcriptional pathways favoring tissue repair. This was demonstrated via significant improvement in voluntary movement parameters (adjusted time mobile, maximum speed, time spent in hut) (Figure 2) and differential gene expression in joint tissues favoring a tissue healing environment with upregulated genes associated with remodeling, cell proliferation and angiogenesis (Figures 3–5). Importantly, all MSC treated groups also showed improved cartilage integrity and synovial scores compared to control animals (Figures 6, 7).

Pattern recognition receptors (PRRs) play a key role in immune and sensory nervous system regulation of pain and inflammation in injury (Rodriguez-Palma et al., 2024). Following tissue damage, PRRs on immune cells are activated to initiate an inflammatory response and sensory neurons concurrently sense these signals PRR expression themselves. In this environment, stimulator of IFN genes (STING) has been recognized as a regulator of pain signaling, where activation of STING receptors has been demonstrated to result in production of type I IFNs in immune cells and sensory neurons and subsequently to elicit antinociceptive effects in neuropathic mice (Donnelly et al., 2021; Sun et al., 2022; Wang et al., 2021; Ishikawa and Glen, 2008). Conversely, deletion of the STING gene has been shown to result in mechanical allodynia (Donnelly et al., 2021). While seemingly counterintuitive to induce production of pro-inflammatory mediators associated with further pain propagation, recent evidence indicates that inflammation can contribute to resolution of pain through further induction of counter-regulatory anti-inflammatory mediators (Sugimoto et al., 2016). Studies exploring the STING pathway elicited type I IFN (INF-α, IFN-β) signaling to alleviate pain have yielded conflicting results, indicating STING agonism and type I IFN production can be both pro- and anti-inflammatory depending on the context in which they are evaluated. For example, STING agonism has produced antinociceptive effects in the central nervous system while STING agonists or recombinant IFN-α injected peripherally have also elicited nociceptive behaviors suggesting pain induction (Liu et al., 2016; Barragan-Iglesias et al., 2020; Fitzgibbon et al., 2019; Franco-Enzastiga et al., 2024). In a rodent model of inflammatory pain, inflammation-induced activation of STING in dorsal root ganglia nociceptors reduced pain-like behaviors (Defaye et al., 2024). Using a gain-of-function STING mutation model, INF-α regulation of voltage-gated potassium channels was identified as the mechanism behind this observed reduction in pain (Defaye et al., 2024). In this study, as the STING-activated MSC themselves likely only persist transiently in the joint, their demonstrated mechanism of action is likely to result from paracrine effects lasting long after the innate immune activated MSC have been cleared. Potential paracrine effects revealed by the transcriptomic studies (Figures 3–5) of joint tissues from animals treated with either TLR3 or STING pathway activated MSC include induction of anabolic processes within joint tissues, suppression of ongoing joint inflammation, and stimulation of joint reparative processes. In addition, cytokines (especially IFNs) secreted by the innate immune activated MSC may have elicited activation of immune counter-regulatory immune responses that further suppressed joint inflammation.

When comparing differentially expressed genes and transcriptomic pathway analyses between treatment groups in this study, treatment of joints with STING pathway activated MSC resulted in upregulation of gene pathways associated with cell cycle activation, proliferation, DNA damage response and repair, and downregulation of genes associated with extracellular matrix, most likely driven by osteoclast, synovial fibroblasts or chondrocytes. Several gene signatures emerged as being potentially particularly relevant to how these two immune pathways (TLR3 and STING pathways) might be functioning to improve joint function. Particularly in the downregulated pathways, significant overlapping gene sets were found between the STING and TLR groups. For example, downregulation of inflammatory and cytokine signaling (biocarta TNFR2, Hallmark IL2 STAT5, Hallmark TNFA, Hallmark inflammatory response) (Supplementary Figure S2); as well as downregulation of cellular stress and senescence (Hallmark hypoxia, p53, UV response and Reactome oncogene induced senescence). Suppression of senescence in fact has been a much-explored area in the reversal of joint damage and treatment of arthritis (Han et al., 2024; Jinjin et al., 2022). HIF1a (Hypoxia-inducible factor-1alpha) and other hypoxia associated genes are also linked to OA related degeneration (Zhang et al., 2025; Zhang and Kong, 2023).

As discussed, IFN-related genes have been previously implicated in the resolution of inflammatory pain (Love et al., 2024), although the role of type I IFNs in this regard remains controversial with reports supporting both pro- and anti-nociceptive actions of IFN-α and IFN-β (Tan et al., 2021). Other potential functions of upregulated IFN pathways in slowing joint degeneration include stimulation of counter-regulatory immune pathways (e.g., IDO pathway, PD-L1 and other checkpoint molecule expression, expansion of Tregs, recruitment of immune suppressive monocytes), all of which may contribute to reducing joint inflammation and preserving cartilage integrity, and suppression of deleterious vascular responses by suppressing abnormal angiogenesis (Rodriguez-Palma et al., 2024). Importantly, a recent publication also highlighted the role of IFN-g in stimulating cartilage regeneration (Kim et al., 2024). Other IFN-regulated pathways may also be involved in improved joint function following injection of activated MSC. For example, alterations in tryptophan metabolism have been previously associated with occurrence and development of OA (Yang et al., 2024). In joints of STING pathway activated-MSC treated mice, one of the most upregulated genes was encoding tryptophan 5-hydroxylase, an enzyme essential to synthesis of the neurotransmitter serotonin. Tryptophan metabolite disturbances have been associated with erosive hand osteoarthritis in people (Binvignat et al., 2023) and conversely, supplementation of tryptophan metabolites including 5-hydroxytryptophan have been shown to suppress inflammation and arthritis through suppression of pro-inflammatory mediator production in a rodent inflammatory collagen-induced arthritis model (Yang et al., 2015). In this study, we observed enhanced mobility in animals treated with immune activated MSC suggesting alleviation of pain, limited structural disease progression in joints evidenced by improved histologic scores (Figures 6, 7). Further investigation into the mechanisms by which induction of inflammatory pathways with STING agonism reduces pain and limits disease progression in the context of OA may lead to new insights to improve our approaches to treat chronic persistent musculoskeletal pain in animals and humans with a variety of different types of OA.

Loss of mobility and articular associated pain are primary reasons for individuals with OA to seek treatment. Therefore, cage monitoring, or “open field testing” of functional activity which we performed here can offer insights as to voluntary behavior and mobility differences relative to baseline as an indication of pain following injury. In this study, by 8 weeks post-DMM surgery, mice injected with two doses of STING-MSC demonstrated increased activity, evidenced by greater adjusted time mobile and less time in their security huts, implying potential differences in clinical or pain responses between groups (Figure 2). Additionally, STING-MSC treated animals exhibited the greatest number of entries to the top of their hut compared to other treatment groups, which reached significance compared to TLR-MSC treated mice at week 6. In this hindlimb injury model, this may indicate an increased willingness to climb, use and propel off the injured limb to enter the top of the hut, potentially indicating earlier return to full hind limb function. It is acknowledged that while cage monitoring or open-field testing in rodent models implies pain following injury, these methods to assess voluntary behavior do not necessarily correlate to pain or mobility experienced by humans. However, the findings reported here do support previous studies in humans where intra-articular cellular based therapies have been reported to decrease pain and improve functionality in knee OA, resulting in increased physical wellness overall (El-Kadiry et al., 2022; Anz et al., 2020).

Histopathology remains the gold standard in animal models to assess structural disease in OA pathology characterized by progressive articular cartilage degradation and osteophyte development (Zaki et al., 2022; Newman et al., 2022; Aigner et al., 2010). In small animal (i.e., rodent) species, evaluation of the entire joint in a single plane allows simultaneous examination of the entire joint, including pathological changes in cartilage degradation, synovial inflammation and fibrosis, meniscal tearing and bone remodeling including marginal osteophytes or enthesiophytes and subchondral sclerosis. Multiple scoring systems have been reported for histopathological evaluation of the mouse knee OA (Glasson et al., 2010; Chambers et al., 1997; Little et al., 2009; McNulty et al., 2011; Sampson et al., 2011; Pinamont et al., 2020; Armstrong et al., 2021), with the destabilization of the medial meniscus (DMM) model presented here representing a reproducible animal model that allows longitudinal evaluation of disease. Haubruck et al. further developed a semi-quantitative mouse OA histopathology scoring system to grade cartilage as well as synovium, meniscus, cruciate ligaments, subchondral bone and marginal osteophytes/enthesiophytes, demonstrating evaluation of a single slide allowed reproducible grading of histopathological changes in joint tissues in this model and reduced scoring time without compromising sensitivity and specificity of results (Haubruck et al., 2023). Using this scoring system, here we notably demonstrated significant improvement in total joint and medial compartment scores with STING pathway activated-MSC treatment compared to control (needle insertion alone) and non-activated MSC, as well as improvement in the lateral compartment between STING pathway activated-MSC to control. When medial and lateral compartments were examined separately, we further demonstrated significant improvement between STING pathway activated-MSC versus control for both medial and lateral tibial and femoral cartilage and synovium scores. TLR3 pathway activated-MSC therapy also resulted in improvement, albeit to a lesser extent compared to STING pathway activated-MSC in overall joint and medial compartment scores compared to needle insertion alone. As animal models remain the foundation for development of therapeutic interventions for human OA with histopathology considered the gold standard for assessment, these findings underscore the potential for immune activated cellular therapy, particularly STING pathway-activated MSC, to mitigate structural disease progression.

Limitations to this study include the relatively short study duration (8 weeks), although this has been reported and considered typical for this model of OA previously. The mouse DMM model was selected as the first in vivo model to initially screen the immune-licensed MSC treatment described due to the low cost of working with rodent models, high reproducibility of the model, fewer/more localized cartilage defects compared to other rodent models (e.g., anterior cruciate ligament transection) making it easier to assess treatment effect, and slower disease progression that more closely mimics human OA, allowing for relatively consistent evaluation of pain-associated behaviors (Glasson et al., 2007; Bapat et al., 2018; Hu et al., 2022). Although significant differences in gait parameters and histopathologic findings were seen between treatment groups by 8 weeks postoperatively, additional information may have been gained by allowing OA to progress for a more prolonged period. Additionally, mice in this study were operated and evaluated following skeletal maturity at 12 weeks of age which would simulate post-traumatic OA in an early adult population (i.e., mid-twenties); however, it is recognized that knee OA in humans frequently occurs in middle age to elderly patients and further evaluation of this treatment strategy at later age time points would be valuable. The lack of sham-operated controls is acknowledged as a study limitation; however, this work was designed to specifically assess acute changes following cell injection and impact of activation procedures rather than further delineating the DMM model.

Furthermore, this study evaluated outcomes following two MSC injections at 3- and 5-weeks post-injury, the timing of which was based upon initial pilot studies (not shown). The MSC used in this study underwent minor phenotypic characterization and it is acknowledged that more detailed MSC phenotyping may be required to sufficiently replicate these data due to heterogeneity in MSC populations and potential variation in isolation protocols between laboratories. Further assessment of injection number, timing of injection(s) during the temporal course of disease progression and dose with an expanded examination of control groups may demonstrate greater improvement and/or provide further information as to the effect of the immune-licensed cellular products. Addition of treatment groups injecting the MSC secretome or extracellular vesicles (EVs) derived from of these immune-activated MSCs were not within the scope of this study; however, evaluation of the impact of immune licensing on immunosuppressive function of EVs or secreted products generated under different culture methods (e.g., 3D vs. 2D monolayer culture) represent future directions of this work. Finally, the use of fetal bovine serum (FBS) in growth media during isolation, culture, and expansion of MSC before immune licensing is acknowledged as a limitation; while the use of FBS in preclinical models is accepted, evaluation of alternate serum-free sources is warranted prior to future translational applications. The findings herein warrant further investigation in additional studies designed to elucidate more specific mechanisms underlying the effect and expanding to large animal models of OA.

In summary, these findings provide key insights into the functional, transcriptomic, and histologic synovial response to intra-articular immune activated (pIC or 2′,3′-cGAMP) activated MSC therapy. Notably, all three MSC therapeutic approaches improved histologic outcomes in this model, while STING-pathway activated-MSC treatment and TLR3-pathway activated MSC resulted in further improvement in key functional parameters and induced transcriptomic changes promoting tissue repair. These findings provide further mechanistic insight as to possible mode of action through which STING and TLR3 pathway activated-MSC may be exerting a therapeutic effect in early post-traumatic OA, relating differential gene expression on the cellular level to improvement seen functionally and structurally on histology. Thus, there is precedent for multiple different IFN pathways being critically involved in reducing joint inflammation and improving mobility and cartilage integrity. These findings suggest that pre-activation of MSC with stimuli that induce strong IFN responses could be an successful strategy to improve the overall effectiveness of cellular therapy for OA.