Ten weeks of high-intensity interval walk training is associated with reduced disease activity and improved innate immune function in older adults with rheumatoid arthritis: a pilot study

Participants

Twelve sedentary participants (11 women and 1 man) aged 64 ± 7 years and with confirmed and stable RA were recruited for the study. Participants were either seropositive or had radiographic joint erosions of finger joints, met the 1987 American College of Rheumatology criteria for RA [33], had no medication changes in the previous 3 months, and were using prednisone ≤ 5 mg/d. Exclusions were known diabetes mellitus or cardiovascular disease and an inability to walk unaided on a treadmill. All participants gave written informed consent, and the study was approved by the Duke University Medical Center Institutional Review Board (IRB no. Pro00064057).

Exercise training

Exercise training consisted of 10 weeks of 3 × 30-minute sessions per week of supervised treadmill walking. In aggregate, participants completed 99% of prescribed exercise sessions. Exercise intensities were determined from a cardiorespiratory fitness test. For exercise prescription, VO₂ reserve was chosen and calculated as previously described [34]. Participants were given between three and six sessions to become accustomed to the exercise (30- to 45-second intervals at target heart rates; total time, 20 minutes). Exercise consisted of a 5-minute warm-up and 5-minute cool-down as part of the total session. Intervals were designed to elicit a heart rate corresponding to 80–90% of VO₂ reserve (high intensity = actual heart rate percentage of 85 ± 5%) and 50–60% VO₂ reserve (active recovery). Speeds did not exceed walking pace (range, 1–4.6 mph), and if heart rate was not achieved by walking speed, gradient (range, 2–15%) was added to increase heart rate. High-intensity intervals were between 60 and 90 seconds, followed by active recovery intervals of a similar duration. Rather than controlling each session for energy expenditure, total intervals per session were adjusted so that the exercise duration per session was 30 minutes. Ratings of perceived exertion were detailed at the end of each high-intensity interval bout.

Assessment of disease activity

All participants were examined by a rheumatologist using previously described methods for determining disease activity using the Disease Activity Score in 28 joints (DAS28) [35]. This score includes assessment of the number of swollen and tender joints, a visual analogue scale for general health, and C-reactive protein (CRP) concentration or erythrocyte sedimentation rate (ESR) (DAS28_CRP and DAS28_ESR, respectively).

Fitness, function, and body composition

Exercise treadmill testing was used to assess cardiorespiratory fitness. Aerobic capacity (VO_2peak) was determined by a graded maximal treadmill test starting at 2 mph/0% grade and then increasing speed and/or grade such that the metabolic demand increased at approximately 3.5 ml/kg/min until volitional exhaustion. Twelve-lead electrocardiogram, ventilation, and gas exchange were continuously assessed and recorded as 15-second averages using a Parvo Metabolic Cart (Parvo Medics, Sandy, UT, USA). Highest 15-second values were used for determination of VO_2peak, and a valid test was confirmed by either a respiratory exchange ratio (RER) > 1.1 (mean RER, 1.2 ± 0.1 at both times) or a rating of perceived exertion ≥ 17. Body composition was assessed according to Siri’s three-compartmental model [36]. Body weight, fat mass, and lean mass were determined by air displacement plethysmography (BOD POD System; COSMED, Rome, Italy) [37]. Resting blood pressure and heart rate were taken following 15 minutes of sitting quietly. Each participant also completed a short battery of physical functioning tests that are indicative of health and frailty in older adults [38–40]. Grip strength was assessed by dynamometry in both hands in triplicate; the best score was taken. Timed Up and Go (TUG), Berg Balance Scale, 30-second chair stands, and 400-m walk tests were all supervised by a trained exercise physiologist. All tests were completed before training and at least 24 hours after the last exercise session.

Physical activity and health questionnaires

During their fasting blood draw visits (see below) at baseline and following the intervention, participants completed standardized validated physical activity and health questionnaires. Physical activity questionnaires were used to determine that participants were not engaging in more physical activity than what was prescribed in the exercise program. Questionnaires used included the Incidental and Planned Exercise Questionnaire (IPEQ), the Stanford Brief Activity Survey (SBAS), the Hospital Anxiety and Depression Scale (HADS), a visual analogue scale for pain (VAS), and the Health Assessment Questionnaire Disability Index (HAQ-DI).

Fasting glucose and insulin

Blood was obtained following a 10-hour overnight fast both before training and between 16 and 24 hours after the last exercise bout. Glucose was measured with a YSI Biochemistry Analyzer (YSI Inc., Yellow Springs, OH, USA), and insulin was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s guidelines (Thermo Fisher Scientific, Waltham, MA, USA); both were measured in duplicate. Hemoglobin A1C (HbA1c) values were obtained through a commercial clinical analysis company (LabCorp, Burlington, NC, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as described previously using the HOMA2-IR online calculator [41, 42].

Immune cell isolation

Complete blood count differentials were measured using a commercial clinical analysis company (LabCorp). Neutrophils were isolated from heparin-treated blood following 2% dextran sedimentation and separation on a discontinuous Percoll gradient as previously described [20]. Neutrophil purity and viability were determined by Giemsa staining (Diff-Quik; Gentaur Europe, Kampenhout, Belgium) and trypan blue exclusion, respectively. Neutrophil purity and viability were consistently ≥ 95%. Neutrophils were resuspended at 3 × 10⁶ cells/ml in either RPMI 1640 (+calcium, +magnesium: RPMI) medium only (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640 containing 0.15% bovine serum albumin (BSA; RPMI-BSA; Sigma-Aldrich) or HBSS+/+ (+ calcium, + magnesium) medium, depending on the assay.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare Life Sciences, Marlborough, MA, USA) density gradient centrifugation. Briefly, blood was diluted 1:1 with RPMI 1640 and layered over the Ficoll before centrifugation at 400 × g for 30 minutes at room temperature. The mononuclear cell layer was removed and washed twice before being resuspended at 1 × 10⁶ cells/ml in RPMI + 1% BSA.

Neutrophil migration

Neutrophil migratory dynamics were assessed using an Insall chamber (Weber Scientific International Ltd., Teddington, UK) as previously described [4, 43]. Briefly, coverslips were coated with 7.5% culture-tested BSA (Sigma-Aldrich), and neutrophils were suspended in RMPI-BSA adhered to this surface for 20 minutes at room temperature. The use of this coating of BSA has previously been shown to mimic the ligand for CD11b and CD18, intercellular adhesion molecule (ICAM)-1 [44]. The coverslip was then inverted on the Insall chamber before addition of buffer (RPMI) alone as a control or buffer containing 100 nM CXCL-8 (R&D Systems, Minneapolis, MN, USA).

Neutrophil migration was monitored using a Zeiss Axiovert 100 inverted microscope (Carl Zeiss Microscopy, Buffalo Grove, IL, USA) fitted with a Hamamatsu ORCA 100 digital camera (Hamamatsu, Japan). Time-lapse recordings and calculations of neutrophil migratory dynamics were performed as previously described [4]. Briefly, the Insall chamber allows the formation of stable chemoattractant gradients, with defined, consistent direction in the y direction for each experiment [43]. Only distance traveled in the y direction over time was included in calculations of chemotaxis. Migration was assessed using three parameters: average cell speed (μm/min) of movement toward the chemokine (termed chemokinesis), average velocity (μm/min) of cells (termed chemotaxis), and accuracy of movement (termed chemotactic index). Chemotactic index is expressed in a comparative scale and arbitrary units (a.u.) ranging from − 1 to + 1. Movement directly toward the chemoattractant is + 1, whereas movement directly away is − 1.

Recordings lasted 20 minutes per experiment, with 20 slides captured using OpenLab software (Improvision, Coventry, UK). The Java software ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used to analyze cell tracks. All analyses were carried out by a single analyst blinded to subject group and cell conditions.

Bacterial phagocytosis

Phagocytosis of opsonized fluorescein isothiocyanate (FITC)-labeled Escherichia coli (Thermo Fisher Scientific) was assessed in whole blood as previously described [20]. Briefly, phagocytosis was assessed in heparin-treated whole blood incubated for 10 minutes at 4 °C (control: no phagocytosis) or 37 °C (test) with FITC-labeled E. coli. Phagocytosis was halted by the addition of cold PBS, whereas cell surface-bound FITC was quenched by addition of 1% trypan blue solution. Unbound free bacteria were removed by washing in PBS, then erythrocytes were lysed and leukocytes were fixed using 1% fix/lyse solution (Thermo Fisher Scientific). Cell DNA was counterstained by addition of propidium iodide (PI) in order to gate on immune cells before flow cytometry was performed, and 10,000 neutrophils were acquired. Median fluorescence intensity (MFI) of the control was subtracted from the test to give a final phagocytic value. The MFI corresponds to a cell-by-cell “amount’ or phagocytic capacity of E. coli.

Neutrophil ROS production

ROS generation was assessed by luminol-amplified chemiluminescence as previously described [45]. Briefly, resting neutrophils [1 × 10⁵ in HBSS+/+ (+ calcium + magnesium)] were dispensed into a 96-well, white, flat-bottomed plate (Corning, Corning, NY, USA) containing 1 μM luminol (pH 7.3; Sigma-Aldrich). Cells were stimulated with 25 nM phorbol 12-myristate 13-acetate (PMA) (test) or HBSS+/+ [46] and immediately assessed for ROS generation at 1-minute intervals for 60 minutes using an Infinite 200 PRO plate reader (Tecan Life Sciences, Männedorf, Switzerland). Experiments were performed in triplicate, with ROS production measured as relative light units and calculated as the AUC.

Measurement of immune cell surface receptor expression

Neutrophil surface receptor expression was assessed in whole blood. Briefly, 100 μl of heparin-treated blood was dispensed into 5-ml tubes and stored at 4 °C in the dark. Cells were stained with anti-CXCR2-phycoerythrin (anti-CXCR2-PE, clone 5E8-C7-F10; Thermo Fisher Scientific), anti-CD16-FITC (clone 3G8; BD Biosciences, San Jose, CA, USA), anti-CD11b-allophycocyanin (anti-CD11b-APC, clone ICRF44; BD Biosciences), anti-CD18-PE (clone 6.7; BD Biosciences), anti-Toll-like receptor 2 (TLR2)-Alexa Fluor 647 (clone 11G7; BD Biosciences), or anti-TLR4-APC (clone HTA-125; Thermo Fisher Scientific), or their relevant concentration-matched isotype controls for 60 minutes on ice in the dark. Following incubation, cells were washed twice in cold PBS, and erythrocytes were lysed and leukocytes were fixed using 1% fix/lyse solution (Thermo Fisher Scientific). Following fixing, cells were washed twice and resuspended in 300 μl of PBS for analysis by flow cytometry.

Monocyte surface receptor expression was assessed on freshly isolated PBMCs (1 × 10⁵ cells/ml). Cells were stained with CD14-Pacific Blue (clone TuK4; Thermo Fisher Scientific), CD16-FITC, TLR2-Alexa Fluor 647, TLR4-PE, anti-HLA-DR-PE-CF594 (clone G46-6; BD Biosciences), or relevant isotype controls for 30 minutes at 4 °C in the dark. Postincubation cells were washed twice in PBS/1% BSA, resuspended in 300 μl of PBS/1% BSA, and transferred to polypropylene fluorescence-activated cell sorting tubes for analysis by flow cytometry.

All flow cytometric analyses were conducted on a BD FACSCanto II (BD Biosciences) flow cytometer equipped with three lasers using the Duke Cancer Institute Core Facility, which maintained daily quality controls of the machine. Ten thousand neutrophils and 5000 monocytes were acquired for analysis. Data were analyzed using FCS Express 6 (De Novo Software, Glendale, CA, USA).

Plasma analyses

Samples were processed immediately for plasma and immune cell isolation, and relevant samples were stored at − 80 °C until analysis. All plasma analyses were completed by the Core Facilities within the Duke Molecular Physiology Institute. Plasma samples obtained following an overnight fast were analyzed for five cytokines and one acute-phase protein: CXCL-8, IL-6, IL-10, TNF-α, IL-1β, and C-reactive protein (CRP). Concentrations of cytokines were determined in duplicate using a human proinflammatory 5-plex sandwich immunoassay according to the manufacturer’s instructions (Meso Scale Discovery, Rockville, MD, USA). High-sensitivity CRP was measured in duplicate using a commercially available ELISA (IBL International, Hamburg, Germany). The lower limits of detection (LLODs) were as follows: CXCL-8 (0.08 pg/ml), IL-6 (0.11 pg/ml), IL-10 (0.05 pg/ml), TNF-α (0.09 pg/ml), IL-1β (0.03 pg/ml), and CRP (0.02 mg/L). All samples had concentrations greater than the LLOD, with the exception of IL-1β with 77% of samples above the LLOD. Plasma concentrations of nonesterified fatty acid (NEFA) were assessed by an enzymatic colorimetric assay on a UniCel DxC600 Analyzer using the manufacturer’s guidelines (Beckman Coulter Life Sciences, Indianapolis, IN, USA).

HIIT effects on health and RA disease activity

HIIT effects on cardiorespiratory fitness and physical function

HIIT effects on systemic inflammation, white blood cell counts and metabolic health

HIIT effects on innate immune function

Improved neutrophil migration and bactericidal function

In response to 10 weeks of HIIT, migration of isolated RA peripheral blood neutrophils toward the chemokine CXCL-8 improved. A doubling of the chemotactic accuracy (chemotactic index; p = 0.003; CI, − 0.3, − 0.1; d = 1.1) was driven by improved directional speed (chemotaxis, p = 0.044; CI, − 1.2, − 0.02; d = 0.66), with no effect on overall speed (chemokinesis, p = 0.272; CI, − 1.5, 0.5; d = 0.33) (Fig. 1a–c). Following HIIT, neutrophils phagocytized more FITC-labeled E. coli (Fig. 2a and b) (p = 0.03; CI, − 2884, − 87; d = 0.71). Accompanying the improved phagocytosis, training enhanced neutrophil PMA-stimulated ROS production (Fig. 2d) (p < 0.001; CI, − 69,964, − 25,737; d = 1.4). No differences were observed for expression of the relevant surface receptors, which could account for these changes, namely the CXCL-8 receptor CXCR2 (Fig. 2e) (p = 0.865; CI, − 105, 90; d = 0.06), the ICAM-1 receptors CD11b/CD18 (Fig. 2f) (p = 0.404; CI, − 96, 207; d = 0.27), TLR4 (Fig. 2g) (p = 0.929; CI, − 158, 147; d = 0.03), or the Fcγ receptor CD16 (Fig. 2h) (p = 0.300; CI, − 571, 1652; d = 0.35). Taken together, these data suggest that HIIT improves neutrophil migratory accuracy and bactericidal functions in patients with stable RA.

Shifts from pro- to anti-inflammatory monocyte phenotypes

Depending on their expression of CD14 and CD16, monocytes can be considered to be composed of three distinct populations (Fig. 3a). Following 10 weeks of HIIT, there were significant reductions in the frequency of total CD16-expressing proinflammatory monocytes (Fig. 3b) (p = 0.002; CI, 2.5, 8.4; d = 1.3) owing to reduced frequency of both intermediate CD14^bright/CD16^positive (Fig. 3c) (p = 0.009; CI, 0.9, 4.7; d = 1.04) and nonclassical CD14^dim/CD16^bright (Fig. 3d) (p = 0.045; CI, 0.1, 5.3; d = 0.73) monocytes. On intermediate monocytes, there was reduced expression of TLR2 (Fig. 3e) (p = 0.005; CI, 182, 738; d = 1.27), TLR4 (Fig. 3f) (p = 0.026; CI, 20, 239; d = 0.90) and HLA-DR (Fig. 3g) (p = 0.037; CI, 306, 7528; d = 0.83). On classical CD14^bright/CD16^negative and nonclassical monocytes, there were no differences for TLR2, TLR4, or HLA-DR expression. Additionally, there was a trend toward an increased percentage of classical monocytes at the expense of proinflammatory, CD16⁺ monocytes (p = 0.062; CI, − 5.7, 0.2; d = 0.68). A greater percentage of classical monocytes may have accounted for increased phagocytosis of FITC-labeled E. coli following training (Fig. 3h) (p = 0.02; CI, − 3260, − 340; d = 0.83). Taken together, these data suggest that HIIT improves the balance of inflammatory pathologically related CD16-expressing monocytes to CD16 negative monocytes.

Disease activity, physical fitness, and function in RA

There is a significant body of literature showing that physical activity and exercise training are safe for patients with RA and do not aggravate disease activity [47–50]. Although studies have suggested that individual components of disease activity can be improved, few have reported actual lowering of disease activity scores [48]. In the present study, using a composite, well-validated disease activity score including assessment of 28 joints for tenderness and swelling, perceived global health, and inflammatory markers (ESR or CRP), overall disease activity was reduced by 38% following training. Reductions appeared to be driven primarily by improvements in joint health, as determined by swollen and tender joints, improved participant feelings of health, and reduced ESR. This reduction was sufficient to reclassify participants on average from moderate to low disease activity. Such a reduction is unlikely to be observed without exercise training; however, because we did not have a control group, this cannot be discounted.

It is not completely clear what accounted for our observed disease activity improvements. One possibility is that by using higher intensities and grade elevations, HIIT had effects on lower extremity strength. Strength improvements were evident by improved sit-to-stand ability. Walking was at faster speeds than individual participants’ normal speeds, as evidenced by comparisons with calculated speeds from both the TUG and 400-m walk speed (data not shown) tests. As such, the only other exercise intervention studies to show disease score reductions incorporated resistance training, suggesting a link between muscle strength and disease activity [51–55]. In light of this, we now add to the literature that HIIT walking is likely associated with improved disease activity. Further, as compared with other aerobic exercise interventions, no significant adverse events and a 9% increase in cardiorespiratory fitness were observed, suggesting that our program is safe and effective in improving cardiorespiratory fitness [30, 32].

HIIT and immune function in RA

Although RA is considered an inflammatory disease characterized by increased systemic and joint inflammation, the pathology of the inflammation is due primarily to a deregulated immune system. Specifically, there is a growing body of evidence suggesting that habitual physical activity and exercise training promote enhanced immune function in both adaptive and innate cells [56]. Although exercise can improve immune function in age-comparable healthy adults [57], few studies have assessed the effects of exercise training on immune function in patients with RA. Because patients with RA are at increased risk of opportunistic infections owing to impaired and reduced immune responses, promoting better immune function likely reduces these risks. Very few studies have assessed immune responses to exercise in RA, with no changes in lymphocyte proliferation or natural killer (NK) cell tumor cell cytotoxicity [31, 58], suggesting that exercise may not enhance primary cell functions. However, because patients with RA are at increased risk of infection and require optimal neutrophil and monocyte function, we were interested in whether HIIT would improve antibacterial functions of innate immune cells.

When compared with healthy age-matched control subjects, patients with stable RA have similar neutrophil migration [59, 60]. Although increased neutrophil migration from the blood to the synovium is observed in the early stages of RA, improved chemotactic accuracy should benefit older patients with RA [4, 59, 61], specifically because poor chemotactic accuracy results in more aberrant tissue damage and elongated resolution of infection and increased inflammatory insult. In a study of healthy older adults, those who completed more steps per day (10,370 ± 3221 vs. 4746 ± 1351) had better neutrophil chemotactic accuracy, suggesting that exercise training may improve neutrophil migratory capacity [20]. Neutrophil migration was also improved in women with hypertension older than 60 years of age following 6 months of exercise training and in young sedentary participants following 2 months of cycling training [62, 63]. We add to the literature by showing, for the first time to our knowledge, that HIIT is associated with improved neutrophil migration toward a common chemokine produced during infectious episodes in older adults with stable RA.

Along with migration, other critical primary neutrophil functions include phagocytosis and ROS killing of pathogens. Although excessive ROS production in the synovium contributes to disease pathology, it is unclear what role blood neutrophil ROS play in RA [5, 64]. Compared with healthy control subjects, in RA blood neutrophil ROS are either similar or reduced [5, 7], whereas reduced ROS with aging is a classic hallmark of neutrophil dysfunction [65]. Similarly, there is no clear consensus regarding whether peripheral blood neutrophil phagocytosis is altered in RA [66–70]. Paino and colleagues suggested that phagocytosis of E. coli is greater in older women with RA than in matched control subjects [67], whereas Turner and colleagues suggested that phagocytosis is reduced in RA [68]. Similarly to our previous HIIT study in healthy adults [25], we show increased stimulated ROS production and increased bacterial phagocytosis following exercise training, suggesting an enhanced ability of neutrophils to kill bacterial pathogens. Furthermore, in agreement with previous studies, functional improvements are not associated with changes in relevant cell surface receptor expression, suggesting intrinsic cellular changes [4, 20, 25].

Neutrophil dysfunction is associated with increased infection risk, and so is monocyte/macrophage dysfunction. Further, with RA and aging, monocytes adopt more of an inflammatory phenotype with the potential to add to the inflammatory milieu. There are three subtypes of blood monocytes with distinct inflammatory and functional characteristics dependent on their expression of CD16 [71–73]. In addition to CD16, TLR2 and TLR4 are typically expressed more on monocytes/macrophages in RA [10, 11, 74]. In healthy sedentary adults, HIIT can reduce expression of CD16, TLR2, and TLR4 while increasing monocyte phagocytosis of E. coli [25, 28]. We add that in older patients with stable RA, HIIT is associated with increased phagocytic capacity of E. coli as well as reductions in the total percentage of CD16-expressing monocytes through reductions in the percentage of both intermediate and nonclassical monocytes. Furthermore, the expression of TLR2, TLR4, and HLA-DR was reduced on the intermediate proinflammatory monocytes, suggesting a reduced proinflammatory phenotype.

HIIT and inflammation in RA

RA medications are aimed at controlling inflammation [75]. Whether exercise training can reduce systemic inflammation in RA is an open question [18–20, 76]. It is likely that exercise has clinically or pathologically meaningful anti-inflammatory effects primarily in individuals with high, uncontrolled inflammation or when exercise is associated with reductions in body fat. The anti-inflammatory effects of exercise training have been reviewed extensively; yet, the mechanisms are still unclear. Long-term training likely exerts most of its anti-inflammatory effects by changing adipocyte mass and function [18]. In the present study, participants had well-controlled stable RA, were taking a range of anti-inflammatory medications, and had minimal nonsignificant changes in body fat; thus, we were not surprised to observe a lack of meaningful changes in systemic concentrations of CRP, IL-1β, IL-6, CXCL-8, IL-10, or TNF-α. These findings are similar to other aerobic-based exercise programs, suggesting that aerobic training without weight loss is insufficient to reduce most inflammatory markers in patients with RA, even those with well-controlled RA [30]. However, in agreement with studies using resistance training, we observed a significant reduction in the inflammatory marker ESR [51]. It is unclear on the basis of our study whether the changes in joint health, as determined by swollen and tender joint count reductions, were associated with reduced joint-specific inflammation. It is possible that joint health improved because of fluid shifts induced by mechanical loading; however, a number of the healthy joints were observed in the hands and arms. To determine relationships with training and inflammation, we conducted correlations between changes in fitness, body fat, and the aforementioned cytokines. No relationships were observed, other than increases in peak VO₂ being associated with increasing concentrations of the anti-inflammatory cytokine IL-10. Future work aimed at understanding relationships between systemic and joint inflammation and exercise training may help illuminate this finding.

Limitations and future directions

Our study has limitations. A lack of an age-matched healthy control group does not allow us to state whether immune function was altered in our study group as a result of their disease. However, a control group in such a population is difficult to select because patients with RA are often more sedentary than healthy control subjects, their immune system shows signs of premature aging, and immune-modifying medications complicate immune function comparisons. In light of this, we could have recruited an RA nonexercise control group in order to show that changes were in fact due to the intervention and not dependent on time. However, a previous exercise study with an RA control group showed that 8 weeks of training was sufficient to increase cardiorespiratory fitness in the exercise group only. Subsequently, the control group did not have changes in immune cell proliferation, NK cell cytotoxicity, or inflammatory cytokine concentrations, suggesting that time alone does not change fitness or alter the immune system and inflammation in patients with RA [31]. Therefore, we are confident that the changes we observed were due to the exercise intervention and not to time alone.

Although this study was a pilot, considerations should be acknowledged regarding the population studied as well as the generalizability and feasibility of a larger, broader RA population to complete such an intervention. Our participants, although representative of our local RA population, were younger and had lower disease activity than much of the larger national RA demographics. As such, it is unclear whether such an intervention would be feasible and provide similar results in an older group with higher disease activity. Further, this was a supervised, structured exercise intervention, which would limit many individuals who are unable to access such expertise and gym facilities. Future studies should address a broader RA population and determine whether this program is feasible and can be transferred to a community- or home-based setting.

Further, we are confident that a significant portion of the effects was due to the intervention and that participants were not engaging in extra exercise outside our study design. Hower, we cannot be certain this did not occur. Although we used validated questionnaires, compared to quantifiable accelerometer data, questionnaires have inherent reliability issues. 

The mechanisms by which exercise training in RA modifies disease activity and joint health or how changes in immune function can contribute are unclear. There is the potential that exercise training can improve immune-mediated joint health by moving dysfunctional cells out of the joint, allowing for replacement with healthier immune cells. Although this hypothesis remains untested in RA, animal exercise models suggest that exercise is associated with a redistribution of immune cells between tissues [77, 78]. Therefore, to elucidate the mechanisms associated with joint improvements, disease activity, and immune function, researchers in future studies should try to compare systemic exercise effects with those in the synovial fluid and local joint tissue.