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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Osteoarthritis Cartilage. 2023 Dec 10;32(3):287–298. doi: 10.1016/j.joca.2023.11.021

Mechanical Loading of Joint Modulates T Cells in Lymph Nodes to Regulate Osteoarthritis

Tibra A Wheeler 1, Adrien Y Antoinette 2, Eshant Bhatia 3,4, Matthew J Kim 1, Chiemezue N Ijomanta 1, Ann Zhao 1, Marjolein C H van der Meulen 1,2,5,*, Ankur Singh 1,2,3,4,6,*
PMCID: PMC10955501  NIHMSID: NIHMS1950615  PMID: 38072172

Abstract

Objective:

The crosstalk of joint pathology with local lymph nodes in osteoarthritis (OA) is poorly understood. We characterized the change in T cells in lymph nodes following load-induced OA and established the association of the presence and migration of T cells to the onset and progression of OA.

Methods:

We used an in vivo model of OA to induce mechanical load-induced joint damage. After cyclic tibial compression of mice, we analyzed lymph nodes for T cells using flow cytometry and joint pathology using histology and microcomputed tomography. The role of T-cell migration and presence of T-cell type was examined using TCRα−/− and an immunomodulatory drug, Sphingosine-1-phosphate (S1P) receptor inhibitor-treated mice, respectively.

Results:

We demonstrate a significant increase in T-cell populations in local lymph nodes in response to joint injury in 10, 16, and 26-week-old mice, and as a function of load duration, 1, 2, and 6 weeks. T-cell expression of inflammatory cytokine markers increased in the local lymph nodes and was associated with load-induced OA progression in the mouse knee. Joint loading in TCRα−/− mice reduced both cartilage degeneration (OARSI: TCRα 0.568, 0.981–0.329 CI; WT 1.328, 2.353–0.749 CI) and osteophyte formation. Inhibition of T-cell egress from lymph nodes attenuated load-induced cartilage degradation (OARSI: Fingolimod: 0.509, 1.821–0.142 CI; Saline 1.210, 1.932–0.758 CI) and decreased localization of T cells in the synovium.

Conclusions:

These results establish the association of lymph node-resident T cells in joint damage and suggest that the S1P receptor modulators and T-cell immunotherapies could be used to treat OA.

Introduction

Osteoarthritis (OA) is a degenerative joint disease that affects approximately 27 million people in the US1–3 and can be induced by several factors including aging, obesity, and joint injury2, 3. Injury to joints can increase the local production of pro-inflammatory mediators of joint tissue damage, which ultimately decreases cartilage structure and function4, 5, however, the rate of disease progression varies considerably6, 7, the causes of which are poorly understood.

Inflammatory mediators, including macrophages and T cells, play a role in OA development and progression8–13. In OA patient biopsies, foci of T lymphocytes and associated cytokines are more pronounced in synovial regions with intense inflammation than in macroscopically noninflamed areas14–16. In peripheral blood samples from OA patients, T cells are increased compared to healthy controls17. T cells also infiltrate the fat pad of OA patients compared to healthy controls18.

The association between lymph nodes, where T cells get activated, and the injured joint remains poorly understood. In post-traumatic OA (PTOA), induced by anterior cruciate ligament transection (ACLT), innate lymphoid cells, γδ+ T cells, and CD4+ T cells contribute to the upregulation of IL-17 expression in the articular compartment and draining inguinal lymph nodes19. IL-17 and immunologically-induced senescence in fibroblasts regulate the response to injury in PTOA. Acute T-cell activation has been demonstrated in a surgical injury model of PTOA20. However, PTOA accounts for only 12% of OA21 and surgery associated with ACTL may evoke an immune response. In contrast, the majority of OA develops progressively from habitual non-traumatic compression loading of joints21. However, the understanding of the connection of mechanical loading of joints with the T cells in local lymph nodes, and their association with the risk of pathology onset and progression is lacking.

We hypothesized that load-induced injury in the joint induces changes to T cells in local lymph nodes, which contributes to the onset and progression of OA through a bi-directional interaction between the lymph node and the affected joint. To test this hypothesis, we used a mouse model of non-traumatic, load-induced OA. In this model, controlled, repeated compression initiates joint damage in cartilage and whole joint pathology without the confounding inflammatory and reparative effects of surgical procedures22, 23. Thus, cartilage matrix damage, including proteoglycan loss, fibrillation and erosion, and osteophyte formation, in this model are caused by mechanical insult to cartilage without disruption of joint structures or joint instability from a traumatic injury present in chemically- and surgically-induced models23–27. Specifically, this noninvasive load-induced model recapitulates OA-like pathology in cartilage and bone after 1, 2 and 6 weeks of daily loading at a peak load of 9N in 10- and 26-wk-old mice23. Using this mouse model, in the present study, we demonstrate an increase in T cells in the local lymph nodes of the limb that was loaded. Using transgenic T-cell knock-out mice, we demonstrated an increase in T-cell subtype associated with OA progression following daily cyclic loading. Using an immunomodulatory agent, Fingolimod that restricts the egress of T cells from lymph nodes, OA progression was attenuated. These findings allude to the possibility of using immunomodulatory therapies to suppress T-cell migration or their absolute numbers to treat OA, an avenue that is yet to be explored.

Materials and Methods

Mice and in vivo mechanical loading

To induce OA, we applied cyclic compression to the left tibia of mice (9.0N-peak load, 1200 cycles, 4Hz, 5 days/week); contralateral limbs served as controls23. We chose to examine young (10-wk-old) and adult (26-wk-old) mice to capture pre-adult and adult ages at which OA-like pathology develops in the cartilage and bone23. 10-wk-old (n=7/group) and 26-wk-old (n=5/group) C57Bl/6 female mice (Jackson Laboratory) were euthanized after one week of daily loading. After initial experiments, 16-wk-old C57Bl/6 female mice (n=5–7/group) were used for the remaining experiments at an intermediate age between 10–26 weeks, when the mice reach peak bone mass28. These mice were euthanized after two weeks of daily loading because our work in female mice shows that two weeks of loading induced cartilage damage, minimal to low-level synovial thickening and cellularity changes, and the majority of osteophytes were cartilaginous29. Supplementary Table 1 outlines all experiments performed. Based on our prior 6-week loading data23, the number of mice per group was determined for our primary outcome of cartilage histological (OARSI) score: n=6 mice/group provides over 90% power to detect a change of 60% with loading (alpha=0.05, standard deviation = 30% of mean). To account for potential tissue losses in harvesting samples, n=7 mice/group was used. A reduced number of samples in the Results reflects the loss of lymph nodes. Power analysis for the flow cytometry data indicated 90% power for n=6/group in 10-wk-old mice. For T-cell knock-out experiments, 16-wk-old TCRα-lacking transgenic C57Bl/6 female mice (TCRα−/−, n=5–7 per group, provided by Brian Rudd, Cornell University) were euthanized after two weeks of loading. TCRα−/− mice are devoid of CD4+CD8- and CD4-CD8+ T cells but have functional γδ T cells30. For flow cytometry, we kept similar numbers of mice. For T-cell trafficking experiments, Fingolimod (FTY720, Cayman Chemical Co), was administered to 16-wk-old female C57Bl/6 mice (n=5–7/group); mice were euthanized after two weeks of daily loading. Fingolimod, an S1P receptor antagonist, limits T-cell ability to traffic through major lymphoid organs31, 32. Mice were randomly divided into treatment groups. Fingolimod (1mg/kg, IP) or saline vehicle (equivalent volume, IP) was administered daily starting 24 hours before loading33, 34. Upon completion of the experimental duration, mice were euthanized by CO2 inhalation. All experimental techniques were approved by the Cornell Institutional Animal Care and Use Committee (Protocols 2007–0025, 2009–0121, and 2017–0059).

Flow cytometry

We only isolated multiple lymph nodes for our initial comparison of the inguinal, iliac, and popliteal responses. Each LN was analyzed separately. For all subsequent studies, the inguinal lymph node was the sole lymph node that we analyzed. Paired comparisons were made between the inguinal lymph nodes from the control and loaded limbs. Dissected lymph nodes were placed in a microcentrifuge tube with 1 mL PBS. Lymph Nodes were degraded by placing them in a 70-μm filter, followed by flowing 1 mL PBS through the filter and mechanically dissociating the lymph nodes using a syringe plunger. After rinsing, cells were transferred to microcentrifuge tubes and centrifuged at 500g for 5 minutes and resuspended in ~200 μL FACS buffer, washed, and stained for cell surface and intracellular markers (Supplementary Table 2). Cells were stained with antibodies at 1:200 dilution for 1 hour at 4°C, using protocols established earlier35–37. The entire volume was analyzed for the total number of cells (BD Accuri C6; BD Biosciences Symphony Analyzer; FlowJo). For cytokine expression, cells were stimulated with a cell stimulation cocktail (TNB-4975, Tonbo Biosciences) for 4.5 hours at 37°C before staining.

Bone morphological changes

Knee joints were harvested and fixed in 4% paraformaldehyde and scanned using microcomputed tomography (microCT) in 70% ethanol at 15μm voxel resolution (μCT35, Scanco; 55 kVp, 145mA, 600 ms integration time), as reported earlier26, 38. Cancellous and cortical bone in the epiphysis and metaphysis of the proximal tibia were analyzed to assess bone morphology. The subchondral bone plate was analyzed for thickness and tissue mineral density (TMD). The cancellous bone in the epiphysis and metaphysis was analyzed for bone volume fraction (BV/TV).

Cartilage degradation

After microCT scanning, tissues were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) and processed for paraffin embedding to analyze cartilage degradation23, 39. Paraffin blocks were sectioned from posterior to anterior at 6-μm thickness using a rotary microtome (Leica RM2255,). Sections were stained using Safranin-O/Fast Green at 90-μm intervals to assess cartilage morphology in the medial tibial plateau. A blinded observer assessed cartilage damage histologically using the modified OARSI scoring system40. Mean OARSI scores were calculated for each limb from multiple scored sections.

Osteophyte formation

To assess osteophyte formation, Safranin-O/Fast Green histological sections were examined by analyzing the medial tibial plateau from three representative sections in the joint (anterior, middle, and posterior). Osteophyte maturity41 was evaluated based on the degree of calcification of ectopic bone. Osteophyte size was based on the medial-lateral width, defined as the maximum distance between the medial end of the epiphysis and the end of the osteophyte; the mean width was calculated for each limb26.

Immunohistochemistry (IHC)

T-cell (CD3+) expression was assessed using IHC. Representative sections from the middle region of the joint were stained. Sections were deparaffinized, rehydrated, incubated in citrate retrieval buffer at 60°C for 60 min, and incubated with 0.3% H2O2 solution for 10 min. Immunostaining was performed using a rabbit-specific HRP/DAB (ABC) IHC detection kit (ab64261, Abcam). Sections were blocked with protein block for 5 min and incubated overnight at 4°C with either rabbit polyclonal anti-CD3 antibody (1:50 dilution, ab5690, Abcam) or rabbit monoclonal negative control anti-IgG (1:50 dilution, ab172730, Abcam). Sections were treated with biotinylated goat anti-rabbit IgG (H+L) for 10 min, followed by treatment with streptavidin peroxidase for 10 min. Finally, sections were incubated with DAB chromogen solution (1:50 dilution) for 5 min, counterstained with hematoxylin for 1 min, and mounted with a coverslip.

Cartilage Mechanics

Local cartilage mechanical properties were measured by nanoindentation. Knee joints were harvested and frozen in PBS-soaked gauze. Limbs were defrosted, and the femur, ligaments, tendons, and meniscus were dissected to expose the tibial cartilage. Tibial plateaus with exposed cartilage then were glued to the surface of a microscope slide (Loctite 409, Henkel Corp.). During indentation, tibial cartilage was maintained in PBS to prevent dehydration. Atomic force microscope (AFM)-based nanoindentation was performed in 16-wk-old female mice (n=3) on the medial and lateral tibial condyles with a spherical tip (Borosilicate particle, 5-μm radius, Au coating, Novascan Technologies) using an AFM (Asylum-MF3D-Bio-AFM-SPM), as previously described 42; indentation was repeated at 8 different locations per tibia.

RNASeq Analysis

Analysis was performed on publicly available data from bulk RNA-sequencing of samples isolated from joint synovial biopsies (data accessible at NCBI GEO database accession GSE89408)43. The details of patient demographics, disease progression status, and recruitment are provided in Guo et al.43. Raw data was log-transformed and differences in gene expression counts of CD4 and CD19 were calculated using one-factor analysis of variance (ANOVA).

Statistical analysis

Lymph node analyses for 10- and 26-wk-old female WT mice were compared using a mixed-effects model with Sidak’s test with factors of age and loading. Histological scores for 10-wk-old female WT mice were compared for the effect of loading using a paired two-tailed t-test. Bar graphs present a mean with 95% CI; individual data are shown for each mouse, and no data are pooled. See Supplementary Data for all raw values and statistical outputs. For Fingolimod and TCRα−/− experiments, the effects of loading and treatment or genotype were determined using a mixed-effects model with fixed effects of loading (control vs. loaded limb) and treatment (saline vs. Fingolimod) or genotype (WT vs. TCRα−/−) and a random effect of mouse with Sidak’s post hoc test for significant interactions. In all statistical analyses, a full interaction between loading and treatment (or genotype) was assessed to estimate the relevance between-group differences. Significance was set at p<0.05.

Results and Discussion

RNA-sequencing of human OA patients validates the presence of immune cells in the joint

To determine whether clinical evidence supports an altered T-cell response in OA patients, we reanalyzed RNA-sequencing data from a public repository (GSE89408) for synovial biopsies from patients with early OA, rheumatoid arthritis, or healthy normal joints43. We observed that the CD4 gene count increased in early OA patients compared to healthy samples (Figure S1). The increase in the CD4 gene in OA patients was similar to that present in the synovial tissues of rheumatoid arthritis patients. In contrast, CD19 gene expression, a marker for B cells, was not different between the early OA and normal tissues and significantly lower than in samples from rheumatoid arthritis patients. These findings indicate that the onset of OA is associated with changes in genes representative of T cells. However, these data cannot establish whether these changes are a direct consequence of mechanical damage to the joints.

T-cell numbers in ipsilateral lymph nodes increased with cyclic tibial compression in female mice

To understand whether T cells in local lymph nodes change due to mechanical loading of the joint, we applied daily loading in 10-wk-old female mice for one week and evaluated lymphocyte counts in lymph nodes using flow cytometry (Figure 1A, Figure S2). Because the knee drains to multiple lymph nodes44, we compared the inguinal, iliac, and popliteal lymph nodes in 10-wk-old female mice after one week of loading. The difference in T cells (Load-Control) was greater in the inguinal lymph node but not significantly from the other locations. T-cell numbers increased in all ipsilateral lymph nodes of loaded limbs, with similar iliac and inguinal responses and lesser popliteal responses, when analyzed with a mixed model (Figure 1B). We focused on the inguinal lymph node because the response was higher in CD3+ and CD3+CD4+ T cells than that measured in the iliac lymph node. The size of the inguinal lymph nodes increased markedly with in vivo loading compared to those in contralateral limbs following one week of daily (5 min) mechanical loading (Figure 1C).

Figure 1. Abundance of T cells increased in inguinal lymph nodes after cyclic tibial compression in female mice.

Figure 1.

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A) Schematic of tibia in loading device and 9N peak load waveform. 10- and 26-wk-old C57Bl/6 female mice underwent 1 week of cyclic tibial compression. Outcomes included flow cytometry of inguinal lymph nodes and histological scoring of joints. Image created using BioRender. B) [Loaded-Control] for the number of CD3+, CD3+CD4+, and CD3+CD8+ T cells in inguinal, iliac, and popliteal lymph nodes of loaded and contralateral limbs after 1-week loading in 10-wk-old female mice. C) Images of inguinal lymph nodes for contralateral (ctrl, top) and loaded (bottom) limbs. D) Gating strategy for number and percentage of T-cell markers after 1-week loading in 10- and 26-wk-old female mice. Number of CD3+CD4+ and CD3+CD8+ T cells in inguinal lymph nodes of loaded and contralateral limbs in female mice. E) (Left) OARSI scores in the medial tibial plateau after 1-week loading in 10-wk-old female mice. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating cartilage erosion; scale bar = 100 μm F) (Left) Osteophyte size in the medial tibial plateau after 1-week loading in 10-wk-old C57Bl/6 female mice. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating osteophyte formation; scale bar = 100μm

We next evaluated the effect of mechanical loading of joints in both young (10-wk-old) and adult (26-wk-old) mice to span ages and match our joint pathology data from prior experiments23. Cyclic compression of the tibia significantly increased the total number of CD3+, CD3+CD4+, and CD3+CD8+ T cells in the inguinal lymph node of loaded limbs in 10- and 26-wk-old female mice, compared to contralateral control limbs that were not subjected to cyclic compressive loading (Figure 1D; Figure S3A). The load-induced increase in lymphocytes after one week was only significant in T cells; CD19+ B cell numbers did not increase in the inguinal lymph nodes compared to controls (Figure S3B).

Concurrent with the increase in T cells in inguinal lymph nodes, we observed significantly more cartilage damage and osteophyte formation in loaded limbs than in controls in 10-wk-old female mice after one week of daily (5 min) mechanical loading (Figure 1E, 1F). This cartilage damage was positively correlated with the number of T cells in loaded limbs and explained 32% of the variation in the OARSI score (Figure S3C). The inguinal lymph nodes from the loaded limb side (n=8) was the sole lymph node that we analyzed. To determine if loading had systemic effects on the number of T cells in contralateral limbs, we analyzed lymph nodes from naïve mice that never received controlled loading. T-cell populations were higher in the lymph nodes of naïve mice than those in mice where contralateral control limbs (not loaded) from experimental mice were analyzed (Figure S3D). The data suggests that loading may have a negative systemic effect on contralateral limbs that requires further in-depth examination in the future. After these initial experiments, we used 16-wk-old mice in the remaining experiments at an intermediate age between 10 and 26 weeks and when the mice reach peak bone mass28.

Pro- and anti-inflammatory cytokine expression increased in T cells with cyclic tibial compression

We characterized the T-cell response in load-induced OA through their expression of cytokine markers, a measure of their activation status. Using flow cytometry, we analyzed the inguinal lymph nodes of 16-wk-old C57Bl/6 female mice following two weeks of cyclic tibial compression (Figure 2). The total number of CD4+ T cells expressing phenotypic markers of IFNγ, TNFα, IL-4, and IL-6 increased significantly in inguinal lymph nodes of loaded limbs compared to controls (Figure 2). However, CD4+IL-10+ and CD4+IL-17+ cell numbers were not different. Both IFNγ and TNFα are inflammatory cytokines produced by helper T cells and expression of IFNγ increases in the synovial tissue and synovium lining of OA patients45. Similarly, with OA, TNFα levels increase in synovial fluid, synovial membrane, cartilage, and subchondral bone46. IL-4 and IL-10 are anti-inflammatory cytokines that are chondroprotective in OA and elevated in the cartilage and synovium of OA patients47, 48.

Figure 2. Both pro- and anti-inflammatory cytokine expression increased with cyclic tibial compression.

Figure 2.

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A) Schematic of experimental design. 16-wk-old C57Bl/6 female mice underwent 2 weeks of cyclic tibial compression. Cytokine expression was measured in inguinal lymph nodes through flow cytometry. Image created using BioRender. B) Number of CD4+IL-4+, CD4+IL-10+, CD4+IL-6+, CD4+IL-17+, CD4+IFNγ+, and CD4+TNFα+ cells in inguinal lymph nodes of loaded and contralateral limbs.

Absence of αβTCR+, but not γδTCR+ T cells, attenuated load-induced cartilage degradation and osteophyte size

To determine whether T cells were involved in the onset and progression of OA, we subjected 16-wk-old TCRα−/− female mice to two weeks of daily in vivo tibial loading and compared to WT counterparts. The total numbers of CD3+, CD3+CD4+, and CD3+CD8+ T cells increased in the inguinal lymph nodes of loaded limbs in WT mice, which is consistent with 10- and 26-wk-old female mice. In contrast, no increase in the number of T cells or T-cell subsets was present in the loaded limbs of 16-wk-old TCRα−/− female mice. However, the total number of γδTCR+ T-cell subsets increased with two weeks of loading in TCRα−/− mice (Figure 3A, 3B, Figure S4A). As expected from our previous data, two weeks of daily loading significantly increased cartilage damage scores in loaded limbs compared to controls in WT mice. In contrast, cartilage damage in loaded limbs of TCRα−/− mice was not different compared to controls (Figure 3C, 3D), suggesting the presence of T cells is associated with the development of cartilage damage. In both groups, osteophytes formed on the medial tibial plateau with loading. Osteophyte maturity was similar in WT and TCRα−/− mice; however, osteophytes in TCRα−/− mice were smaller than those in WT mice, further suggesting a role of T cells in osteophyte growth (Figure 3E, 3F; Figure S4B). The decreased osteophyte formation in these mice could reflect the altered skeletal phenotype of this mouse strain and needs to be confirmed by examination of bone structure differences between TCRα−/− mice and their WT controls. In these studies, any changes in the synovium/joint capsule were not assessed but may be evaluated in the future.

Figure 3. The absence of αβ T cells attenuated load-induced cartilage degradation and osteophyte size.

Figure 3.

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A) Schematic of experimental design. 16-wk-old C57Bl/6 and TCRα−/− female mice underwent 2 weeks of cyclic tibial compression. Outcomes included flow cytometry of inguinal lymph nodes and histological scoring and microCT of joints. Image created using BioRender. B) Number of CD3+CD4+, CD3+CD8+, and γδTCR+ T cells in inguinal lymph nodes of loaded and contralateral limbs. C) OARSI scores in the medial tibial plateau. D) Safranin-O/Fast Green images of contralateral and loaded limbs indicating cartilage erosion; scale bar = 100 μm E) Osteophyte size in the medial tibial plateau. F) Safranin-O/Fast Green images of the contralateral and loaded limbs indicating osteophyte formation; scale bar = 100 μm G) Schematic of AFM-nanoindentation on tibial cartilage. H) Cartilage indentation stiffness. I) MicroCT analysis of subchondral bone plate thickness and TMD and epiphysis and metaphysis BV fraction.

To determine changes in micrometer level stiffness of cartilage tissue as a function of T-cell presence, we performed nanoindentation studies on loaded limbs from WT and TCRα−/− mice. Cartilage stiffness was reduced in loaded limbs compared to controls of WT and TCRα−/− mice. However, overall cartilage stiffness was reduced less with loading in TCRα−/− than WT mice as measured by nanoindentation on the tibial condyle cartilage surfaces (Figure 3G, 3H), suggesting microdamage may be evident in the joint by nanoindentation prior to observing histological joint tissue damage.

Reduced subchondral bone mass is associated with early-stage OA49. Subchondral bone plate thickness and TMD or epiphyseal cancellous BV/TV did not change following loading in WT mice vs. TCRα−/− mice. However, WT mice had overall thicker subchondral bone plates and metaphyseal cancellous BV/TV compared to TCRα−/− mice when both contralateral and loaded limbs were pooled (Figure 3I). The subchondral and epiphyseal bone measurements suggest a skeletal phenotype in TCRα−/− mice. While a complete skeletal phenotype assessment was not performed, previously in 14-week-old female mice, tibial areal BMD was lower in TCRα KO mice compared to WT measured by dual-energy x-ray absorptiometry, but cancellous bone volume fraction (BV/TV) and cortical area of the tibia were not different when measured by microCT50. TMD and epiphyseal cancellous BV/TV were not different between genotypes. Unlike the subchondral plate, loading increased metaphyseal cancellous BV/TV, but only in TCRα−/− mice. The lower metaphyseal cancellous bone volume fraction in TCRα−/− mice suggested that load-induced OA pathology would be more severe in this mouse model, which our data did not support. Even with thinner subchondral bone plates and lower metaphyseal BV/TV, TCRα−/− mice had improved cartilage health after loading. The mechanotransduction in the cartilage and bone may be differentially altered in the TCRα−/− mice and could be evaluated in the future.

Restricted in vivo T-cell egress from lymphoid tissues reduced OA pathology with cyclic tibial compression

Knockout of T cells eliminates not only T cells but also prevents the secretion of cytokines in lymph nodes that may be involved in the onset and progression of OA through a systemic effect. To determine whether the egress of specific T-cell subsets in inguinal lymph nodes of loaded joints was involved in the onset and progression of OA, we treated mice with an inhibitor of the S1P receptor that restricts the egress of T cells from lymphoid tissues. Here we used Fingolimod, a small molecule inhibitor that after phosphorylation, binds to S1P1 receptors on T cells and disables the responsiveness of T cells to the egress signal S1P51, 52.

In saline-treated 16-wk-old C57Bl/6 female mice, two weeks of loading increased lymph node size compared to control limbs (Figure 4A, 4B). Fingolimod treatment alone increased the lymph node size in both control and loaded limbs compared to lymph nodes in contralateral limbs of saline-treated mice, likely because of the restricted egress of T cells. However, lymph node size in loaded Fingolimod-treated mice was not significantly different compared to contralateral limbs. Two weeks of cyclic tibial compression increased specific T-cell subsets in loaded limbs of saline-treated mice compared to control limbs. Specifically, the total numbers of CD3+, CD3+CD4+, and γδ T-cell receptor (TCR)+ T-cell subsets increased in the inguinal lymph node of loaded limbs of saline-treated mice (Figure 4C). Within the CD4+ T-cell subsets, loading increased the number of CD3+CD4+FoxP3+ (Treg) in saline-treated mice but not in Fingolimod-treated mice (Figure 4C). In contrast, neither saline nor Fingolimod treatment changed CD3+CD4+GATA3+ (TH2) and CD3+CD4+Tbet+Rorγt+ T-cell (TH17) subsets in inguinal lymph nodes (Figure 4C; Figure S5A). This was an unexpected finding as we anticipated an increase in T cells in response to loading which would be further increased in the presence of the drug that prevents cells from leaving the lymph node. However, in previous reports, a high dose of fingolimod (1 mg/kg) reduced the frequency of T cells in the inguinal lymph node53, 54. In saline-treated mice, cartilage damage increased in the loaded limb compared to contralateral controls. With Fingolimod treatment, cartilage damage scores in loaded limbs remained low and were not different than contralateral controls (Figure 4D), suggesting that T-cell egress was related to cartilage degradation in load-induced OA.

Figure 4. Fingolimod treatment attenuated load-induced cartilage degradation after 2 weeks of cyclic tibial compression.

Figure 4.

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A) Schematic of experimental design. 16-wk-old C57Bl/6 female mice underwent 2 weeks of cyclic tibial compression with daily saline or Fingolimod injections. Outcomes included flow cytometry of inguinal lymph nodes and histological scoring and microCT of joints. Image created using BioRender. B) Images of inguinal lymph nodes of contralateral and loaded limbs for vehicle and Fingolimodtreated groups C) Number of CD3+CD4+, CD3+CD8+, γδTCR, CD3+CD4+FoxP3+, and CD3+CD4+GATA3+ T cells in inguinal lymph nodes of loaded and contralateral limbs. D) (Left) OARSI scores in the medial tibial plateau. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating cartilage erosion; scale bar = 100 μm E) (Left) Osteophyte size in the medial tibial plateau. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating osteophyte formation; scale bar = 100 μm F) MicroCT analysis of epiphysis and metaphysis BV fraction. G) IHC images indicating reduced CD3+ T-cell staining in Fingolimod-treated limbs; scale bar=100μm

Osteophyte size and maturity in both saline- and Fingolimod-treated mice were similar on the medial tibial plateau after two weeks of loading and greater than those of controls (Figure 4E; Supplementary Figure S5B). Although loading did not alter subchondral bone plate thickness or TMD in either treatment group, Fingolimod treatment itself increased TMD in both loaded and control limbs compared to saline-treated treatment (Supplementary Figure S5C). Similarly, metaphyseal and epiphyseal cancellous BV/TV was higher in Fingolimod-treated than saline-treated mice but not affected by loading with either treatment (Figure 4F). In saline-treated mice, CD3+ T cells were present in the osteophyte and synovium of loaded joints. In contrast, we observed fewer T cells in tissues from Fingolimod-treated mice (Figure 4G). Taken together, these findings indicate that restricting the egress of T cells from lymph nodes through S1P receptor modulation can reduce OA pathology induced with cyclic tibial compression. While the primary role of Fingolimod is restricting the egress of T cells, recent studies in autoimmunity have further demonstrated a direct anti-inflammatory effect of Fingolimod on T-cell populations and improvement of peripheral tolerance mechanisms54, which remains to be investigated in current OA setting.

Helper and γδ T cells in ipsilateral lymph nodes increased with long-term cyclic tibial compression

To determine whether the T-cell response persists with longer-duration loading, we loaded female WT mice for six weeks. After six weeks of daily loading, the number of CD3+CD4+ and γδTCR+ T-cell subsets remained significantly elevated in lymph nodes of loaded limbs compared to controls of 16-wk-old female mice (Figure 5A, B). The CD3+ and CD3+CD8+ T-cell subsets also showed an increase but were not statistically significant, and the variability alludes to a potential biological effect. Consistent with the T-cell response, cartilage damage and osteophyte formation increased in loaded limbs after six weeks of loading (Figure 5C, D). At this longer load duration, the increase in the CD4+ T-cell population was consistent with the fact that many cytokines produced by CD4+ T cells are implicated in the joint damage associated with OA. Collectively, these studies suggest that the T-cell response is not short-lived and remains active with long-term loading. γδTCR+ T cells also still were elevated at this time point; however, the exact role of γδTCR+ T cells in OA pathology remains unclear and requires further investigation.

Figure 5. T helper and γδ T cells in ipsilateral lymph nodes increased with prolonged cyclic tibial compression in female mice.

Figure 5.

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A) Schematic of experimental design. 16-wk-old C57Bl/6 female mice underwent 6 weeks of cyclic tibial compression. Outcomes included flow cytometry of inguinal lymph nodes and histological scoring of joints. Image created using BioRender. B) Number of CD3+, CD3+CD4+, CD3+CD8+, γδTCR+ T cells in inguinal lymph nodes of loaded and contralateral limbs. C) (Left) OARSI scores in the medial tibial plateau. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating cartilage erosion; scale bar = 100 μm D) (Left) Osteophyte size in the medial tibial plateau. (Right) Safranin-O/Fast Green images of contralateral and loaded limbs indicating osteophyte formation; scale bar = 100μm

Conclusion

We have demonstrated an association of CD4+ T cells in cartilage degradation and the progression of load-induced OA. Our findings indicate that upon damaging loading of the knee joint in mice, T cells increased in local lymph nodes, followed by the development and progression of OA-like pathology in the knee. When the CD4+ TCRα−/− mice were used, OA progression was reduced. In contrast, when the T-cell egress was restricted from the lymphoid tissues, the cartilage damage in response to loading of the joint was reduced; however, osteophytes still formed. Based on these findings, we propose a model (Figure 6) of T-cell regulation in the development and progression of OA.

Figure 6. Schematic summarizing the hypothesized effects of altering T-cell presence on OA development.

Figure 6.

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Summary of the effect of normal T-cell levels on cartilage and osteophyte formation in load-induced OA development. Despite different effects on osteophyte formation, the absence of T cells (via TCRα−/−) and the restricted migration of T cells (via Fingolimod treatment) both reduce cartilage damage. Image created using BioRender.

The damage in this loading model depends on many factors, including animal sex and age, load magnitude, and load duration. In the ATCL model, CD4+ T cells increased IL-17a protein expression in the inguinal lymph node, whereas IFN-γ and IL-4 did not change19. This report is in contrast to our results in which mechanical loading increased IFN-γ and IL-4 but not IL-17 in T cells. T, the contrasting results could also be attributed to potential differences in the B6 mouse source. The altered joint kinematics with injury25, lack of surgery and other factors such as animal age and sex could account for differences between the results. More studies are required to understand the T-cell response and the role of the immune system in different joint damage models and the gender- and age-dependency of the T-cell response. In future studies, analysis of peripheral blood, synovium, and synovial fluid following mechanical loading could provide deeper insights. The balance between effector and cytotoxic T cells was not reported or considered in the interpretation of the data. Furthermore, the TCRα−/− mouse model only lacks alpha beta T cells, and Fingolimod affects all T cells. Specifically depleting CD4 or CD8 T cells would elucidate T-cell subtype-specific roles in OA pathogenesis and enable the use of S1P modulators and other T-cell immunomodulators as potential therapeutics for OA treatment. Lastly, the results demonstrated in this study are only thoroughly investigated in female mice, and while preliminary studies with male mice showed T cell increase with loading, a detailed study addressing sex as a biological variable needs to be performed.

Supplementary Material

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Acknowledgments

We thank Dr. Lionel Ivashkiv and Hayat Ben Larbi at the Hospital for Special Surgery, Simon Ortiz, Carolyn Chlebek, Amanda Rooney, Tyler McNeill, Kristine Lai, Dr. Brian Rudd and his laboratory, Dr. Glenn Jackson, and the Center for Animal Resources and Education (CARE) staff at Cornell University. We acknowledge partial financial support from the 3M Non-Tenured Faculty Award (AS), National Institutes of Health (R01-AI132738, AS), National Institutes of Health (R21-AR064034, MvdM), Department of Defense (USAMRMC W81XWH-17-1-0540, MvdM), National Science Foundation (#1605935, MvdM), National Science Foundation Graduate Research Fellowship (DGE-1650441, TAW), and the Cornell Sloan and Colman Diversity Fellowships (TAW).

Footnotes

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Data Availability Statement

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1950615-supplement-1.pdf (797.1KB, pdf)
2
NIHMS1950615-supplement-2.docx (85.3KB, docx)
3
NIHMS1950615-supplement-3.xlsx (36.2KB, xlsx)

Data Availability Statement

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

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