Influence of routine exercise on the peripheral immune system to prevent and alleviate pain

Highlights • In rodents routine exercise prevents and reduces pain by altering the immune system.• In rodents regular exercise decreases pro-inflammatory cytokines and immune cells.• In rodents routine exercise increases anti-inflammatory cytokines and immune cells.• In humans regular exercise may promote a systemic anti-inflammatory immune profile.


Introduction
Physical inactivity increases all-cause mortality and is a risk factor for the development of obesity, cardiovascular disease, diabetes, cancer, dementia, and depression (Biswas et al., 2015;Fischer et al., 2007;Lahart et al., 2015;Nocon et al., 2008;Paffenbarger et al., 1994;Rovio et al., 2005;Tuomilehto et al., 2001;Wolin et al., 2009). Physical inactivity is also a risk factor for the development of chronic pain conditions (Landmark et al., 2011;Landmark et al., 2013). Repeated exercise as an intervention is commonly prescribed for conditions such as low back pain, osteoarthritis, and fibromyalgia with moderate to strong evidence of effectiveness due to favorable effects in reducing pain severity and improving physical function, cognition, sleep, mental health and more (Bidonde et al., 2017;Geneen et al., 2017;Qaseem et al., 2017;Busch et al., 2011;Busch et al., 2013). To understand how routine exercise prevents and alleviates pain; research has been carried out to identify mechanisms of exercise induced analgesia. This research has demonstrated that exercise has several systemic effects that reduce pain at the level of the brain, spinal cord, and in the periphery and have been reviewed elsewhere (Lesnak and Sluka, 2020, Lima et al., 2017. There has been a recent surge in research surrounding the role of the peripheral immune system in the development and maintenance of chronic pain (Laumet et al., 2019, Totsch and Sorge, 2017, Tan et al., 2021, Baral et al., 2019. In preclinical research, genetic strains that limit immune system function and immune cell depletion techniques have implicated a role of the immune system in pain development. Athymic or Rag1-/-mice lack functional T cells and have reduced pain hypersensitivity following induction of neuropathic pain (Laumet et al., 2019). Similarly, depletion of macrophages from the gastrocnemius muscle prior to induction of pain prevents the onset of muscle hyperalgesia and depletion of neutrophils attenuates the development of paw hyperalgesia following paw incision (Carreira et al., 2013;de Azambuja et al., 2021;Gong et al., 2016;Gregory et al., 2016). In individuals with chronic pain, there is often increases in circulating pro-inflammatory cytokines and involvement of the immune system in disease etiology has been implicated in fibromyalgia, osteoarthritis, rheumatoid arthritis, peripheral neuropathy, low back pain, and complex regional pain syndrome (Alivernini et al., 2020, Held et al., 2019, Ernberg et al., 2018, Teodorczyk-Injeyan et al., 2018, Bäckryd et al., 2017, Hysing et al., 2019, Totsch and Sorge, 2017, Merriwether et al., 2021. Upstream mediators suggest ATP, lactate, and hydrogen ions activate receptors (P2X4, P2X7, ASIC3) on macrophages to induce release of inflammatory cytokines and enhance pain Oliveira-Fusaro, 2020). Therefore, researchers have explored if repeated exercise modulates the peripheral immune system to produce analgesia. Once again, repeated exercise produces systemic changes throughout the body by altering the immune systems response to pain in both preclinical and clinical research. The purpose of this review is to summarize the findings on routine exercises role in modulating the peripheral immune system to prevent or alleviate pain.

Animal studies
Animal models are commonly used to study mechanisms of exercise induced analgesia. These models frequently use aerobic forms of regular exercise such as treadmill training, swimming, or voluntary wheel running (Lesnak and Sluka, 2020), however recently a resistance training model was developed which was effective at preventing muscle pain (Lesnak et al., 2022). The following papers discussed in this review, all found that routine exercise was able to prevent or reduce hyperalgesia in various pain models including inflammatory, muscle, neuropathic, and osteoarthritic. In summary, regular exercise modulates immune response to pain at the multiple sites, including at site of injury, in the dorsal root ganglia (DRG), and systemically throughout the body (Table 1).

Site of injury or pain model induction
The most extensive research on exercises ability to modulate the peripheral immune system has been done at the site of injury or pain model induction. This work demonstrates routine exercise can impact swelling, physiological changes initiated by pain model, number and phenotype of local immune cells, and presence of local cytokines in response to pain model induction (Fig. 1).

Swelling
Exercise shows mixed results when it comes to its ability to prevent or alleviate swelling after induction of pain. Injection of Complete Freund's adjuvant (CFA) into the hindpaw of rodents produces a robust immune response resulting in swelling of the limb and mechanical hypersensitivity. Voluntary wheel running performed after injection of CFA causes no significant reduction in swelling in rats (Pitcher et al., 2017); however, in mice, swimming initiated after injection of CFA results in lower levels of edema when compared with sedentary animals (Ludtke et al., 2020). Interestingly rats with CFA hindpaw injections given access to voluntary wheel running, ran comparable distances as rats who received CFA vehicle injections suggesting the lack of reduction in edema was not caused by a lack of running wheel activity induced by CFA. Voluntary wheel running initiated after induction of a tibial fracture pain model does not reduce temperature or edema of the injured hindpaw (Shi et al., 2018). Lastly, treadmill training for two weeks prior to induction of a gout model, reduces the amount of ankle swelling in mice compared with sedentary animals (Jablonski et al., 2020). The conflicting results regarding the impact of exercise on swelling could be due to the pain model studied, species utilized, and exercise parameters such as type and volume. Despite the lack of reduction in edema in the cited studies, exercise was still shown to reduce hindpaw hypersensitivity and weightbearing asymmetry in response to CFA (Pitcher et al., 2017) and tibial fracture (Shi et al., 2018) suggesting analgesic effects of exercise are not solely produced through a reduction in swelling.

Progression of physiological changes initiated by pain models
Routine exercise slows or prevents disease progression in various animal pain models. Models of knee osteoarthritis are induced either through intraarticular injection of an insult such as monosodium iodoacetate (MIA), 3% kaolin and carrageenan or by anterior cruciate ligament (ACL) transection. These OA models cause progressive joint pathology demonstrated through reductions in cartilage thickness, structural alterations in the synovium, and trabecular bone loss. Treadmill training initiated 10 days after MIA injection reduces the amount of trabecular bone loss in rats (Allen et al., 2015). While treadmill training initiated 2 weeks after ACL transection slows cartilage degeneration in rats (Castrogiovanni et al., 2019).
Muscle pain models applied at the gastrocnemius muscle are induced by intramuscular injections of insults such as acidic saline or carrageenan, through fatiguing muscle contractions, or by long term ankle immobilization. Induction of muscle pain causes a robust immune response at the muscle level demonstrated through both transcriptional alterations and increases in immune cells (Gong et al., 2016;Gregory et al., 2015;Lesnak et al., 2022). RNA sequencing performed on the gastrocnemius muscle 24 hours after induction of activity-induced muscle pain demonstrates upregulation of several immune system pathways including chemokine signaling, viral protein interaction, tolllike receptor signaling, and NOD-like receptor signaling in both sedentary male and female mice. This work also reveals a female specific activation of the antigen processing and presentation pathway in sedentary females only. Eight weeks of voluntary wheel running prior to induction of muscle pain causes a dampening of the transcriptional immune system response in females but not males, with a complete prevention in the transcriptional activation of the antigen processing and presentation pathway (Lesnak et al., 2022). This suggests repeated exercise performed prior to muscle pain can blunt the immune system response to later insults to prevent pain.
Lastly, chronic constriction injury (CCI) or a crush of the sciatic nerve is routinely used as a model of neuropathic pain in rodents and results in significant damage to the sciatic nerve and subsequent hindpaw mechanical hypersensitivity. Treadmill training performed 2 weeks prior and 3 to 7 days post CCI causes an accelerated regeneration of the injured sciatic nerve demonstrated through higher amounts of growth associated protein 43 and Cdc2 in the sciatic nerves of mice (Cobianchi et al., 2010). Similarly, treadmill training initiated after sciatic nerve crush results in a higher number of myelinated fibers which also demonstrated increased fiber diameter and myelin sheath thickness in mice (Bobinski et al., 2011).

Immune cell numbers and phenotype
Routine exercise alters the amount and phenotype of immune cells at the site of induction of the pain model. Induction of muscle pain produces an increase in the number of macrophages in the gastrocnemius muscle (de Azambuja et al., 2021;Gong et al., 2016;Gregory et al., 2015;Honda et al., 2022;Lesnak et al., 2022). Swimming prior to carrageenan injection attenuates the number of macrophages in the gastrocnemius muscle of mice (de Azambuja et al., 2021); however, voluntary wheel running prior to activity-induced muscle pain has no impact on the number of macrophages in the gastrocnemius (Lesnak et al., 2022). Voluntary wheel running prevents an increase in CD3 T cells in the gastrocnemius muscle in response to activity-induced muscle pain in male and female mice (Lesnak et al., 2022). Lastly, electrically stimulated muscle contractions initiated after joint immobilization decreases the number of macrophages in the gastrocnemius muscle of rats when compared with sedentary animals (Honda et al., 2022).
Macrophages are plastic and can have a pro-inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2) (Stout et al., 2005). M1 macrophages are responsible for phagocytic activity and secrete proinflammatory cytokines such as IL-1β, IL-6, and TNFα, while M2 macrophages are responsible for tissue repair and secrete anti-inflammatory cytokines such as IL-10, IL-4 and IL-1ra (Stout et al., 2005;Mosser and

Reference
Pain  (Ikeda et al., 2013;. In sedentary animals, induction of muscle pain through either carrageenan injection or fatiguing muscle contractions combined with acidic saline increases M1 macrophages (de Azambuja et al., 2021;Lesnak et al., 2022). However, animals with prior swimming or voluntary wheel running see a predominant increase in M2 macrophages in the gastrocnemius muscle after induction of the pain model (de Azambuja et al., 2021;Lesnak et al., 2022). Again, this suggests that prior exercise can impact how the immune system responds to later insults to prevent the onset of muscle pain. Similar results are seen in models of neuropathic pain induced via sciatic nerve injuries. In sedentary animals, there are increases in the number of macrophages in the sciatic nerve with a higher predominance of the M1 phenotype following nerve injury (Bobinski et al., 2018;Grace et al., 2016). Voluntary wheel running performed prior to sciatic nerve injury decreases the total number of macrophages and increases the proportion of M2 macrophages in the sciatic nerve (Grace et al., 2016). Treadmill training initiated 3 days after sciatic nerve injury causes a shift in macrophage phenotype in the sciatic nerve at the site of injury toward a higher prevalence of M2 macrophages but did not reduce total number of macrophages (Bobinski et al., 2018). This shift in macrophage phenotype is prevented in IL-4 knockout mice suggesting exercises ability to promote M2 macrophages is mediated through release of IL-4 (Bobinski et al., 2018).
Routine exercise alters local immune cell quantities in less studied pain models. Treadmill training performed prior to induction of a gout model decreases the number of macrophages and neutrophils into the ankle joint synovium of mice (Jablonski et al., 2020). In a rat model of endometriosis, voluntary wheel running two weeks prior and after model induction results in decreased numbers of mast cells and macrophages in vesicles and mesenteric fat (Appleyard et al., 2021). Voluntary wheel running attenuates the number of mast cells in the bladder and prostate in mice with perigenital hypersensitivity produced by neonatal maternal separation (Fuentes et al., 2021). Lastly, in a knee OA model induced by intraarticular injection of 3% kaolin and carrageenan, quadriceps muscle stimulation initiated after induction of the pain model reduced the number of macrophages in the knee joint synovium in rats compared with sedentary animals (Ishikawa et al., 2019). Thus, these data consistently shows regular exercise modulates the immune system in uninjured animals, and after induction of pain in a variety of animal models.

Alterations in cytokines
Regular exercise can also alter the balance of pro and antiinflammatory cytokines at the site of injury. Most commonly studied is the ability of exercise to reduce pro-inflammatory cytokines IL-1β, TNFα, and IL-6 and increase the levels of anti-inflammatory cytokines IL-4, IL-10, and IL-1ra. In a mouse model of muscle pain produced by limb ischemia and reperfusion, prior voluntary wheel running prevents increases in IL-1β in forepaw muscles (Ross et al., 2010). Following carrageenan injection into the gastrocnemius muscle there are increases in pro-inflammatory cytokine IL-1β in the muscle which is prevented by prior swimming exercise (de Azambuja et al., 2021), and swimming exercise increases the anti-inflammatory cytokine IL-10 in the muscle (de Azambuja et al., 2021). IL-10 seems to be a crucial mediator in exercises ability to prevent muscle pain as the analgesic effects of voluntary wheel running prior to muscle pain induction is prevented by IL-10 antibody treatment and local intramuscular delivery of IL-10 attenuates the onset of muscle pain . Similarly, a single eccentric exercise bout increases IL-10 in the gastrocnemius muscle and knockdown of IL-10 receptors in the DRG blocks exercises ability to prevent muscle pain to a subsequent overuse exercise session (Alvarez et al., 2017).
Similarly, in models of neuropathic pain induced by sciatic nerve injury or diabetic neuropathy, sedentary animals show an increase in pro-inflammatory cytokines IL-1β, TNFα, and IL-6, and a decrease in anti-inflammatory cytokines following induction of pain in the injured nerve (Bobinski et al., 2011;Chen et al., 2012;Chen et al., 2014;Huang et al., 2017;Tsai et al., 2017). When treadmill training is performed prior to sciatic nerve injury, there are decreases in pro-inflammatory cytokines IL-1β and TNFα in the injured nerves (Bobinski et al., 2011). When initiated after induction of neuropathic pain, treadmill training and swimming both reduce IL-1β, TNFα, and IL-6 in the injured nerves (Chen et al., 2012;Chen et al., 2014;Huang et al., 2017;Tsai et al., 2017) and increases the anti-inflammatory cytokines IL-10, IL-4, and IL-1ra in the injured nerve (Bobinski et al., 2018;Chen et al., 2012;Huang et al., 2017;Tsai et al., 2017). Blockade of IL-4 via IL-4 knockout mice or treatment with an IL-4 antibody, prevents the analgesic effects of treadmill training (Bobinski et al., 2018). This suggests increases in antiinflammatory cytokines, particularly IL-4, within the injured nerve appear to mediate the ability of regular exercise to alleviate neuropathic pain.
In an animal model of gout, prior treadmill training attenuates the increase in IL-1β in the ankle joint synovium of mice (Jablonski et al., 2020). In knee OA produced by ACL transection, treadmill training initiated after injury reduces levels of IL-1β and TNFα and increases levels of IL-4 and IL-10 in the knee joint synovium of rats  (Castrogiovanni et al., 2019). In a mouse model of intervertebral disc degeneration, voluntary wheel running results in decreased levels of IL-1β and TNFα in the multifidus muscle (James et al., 2018). Lastly, voluntary wheel running initiated after a tibial joint fracture reduces levels of IL-1β and CCL2 in hindpaw skin of mice (Shi et al., 2018). In sum routine exercise demonstrates the ability to alter local immune response to pain when performed before or after the insult. Exercise modulates the local response by reducing swelling, slowing disease progression, decreasing numbers of immune cells, and by shifting the local environment to an anti-inflammatory setting through manipulation of macrophage phenotype and presence of cytokines.

Dorsal root ganglia
Repeated exercise also modulates the immune system at the level of DRG through altering the presence of immune cells, attenuating the number of neurotrophic and transcription factors, normalizing functional activity, and reducing pro-inflammatory cytokines (Fig. 1). The Fig. 1. Summary of the impact of regular exercise on the peripheral immune system to prevent and alleviate pain. Exercise works at the site of injury, dorsal root ganglia, and systemically to decrease pro-inflammatory macrophages and cytokines and increase anti-inflammatory macrophages and cytokines.
DRG serves as the cell body for nociceptors in the periphery which carry sensory information to the spinal cord, and thus serves as an integral location for studying pain mechanisms. First, exercise attenuates the number of macrophages in the DRG in animal models of neuropathic pain. Voluntary wheel running prior to CCI of the sciatic nerve reduces macrophage accumulation in the DRG of rats (Grace et al., 2016). Similarly, a model of neuropathic pain induced by a unilateral C5 contusion results in increased macrophages in the DRG of rats and treadmill training performed afterwards, reduces the number of macrophages (Chhaya et al., 2019).
Next, regular exercise reduces the presence of neurotrophic and transcription factors in the DRG. Voluntary wheel running prior to CCI of the sciatic nerve attenuates increases in the transcription factor ATF3 in rats (Grace et al., 2016). Treadmill training and swimming initiated after sciatic nerve ligation reduces DRG levels of neurotrophic factors NGF and BDNF in response to sciatic nerve ligation in mice and rats (Almeida et al., 2015;Lopez-Alvarez et al., 2015). Finally, treadmill training reduces levels of substance P in the DRG following induction of post-incisional pain in rats .
Routine exercise modulates expression levels of ion channels and receptors on DRGs. In rats with diabetic induced neuropathic pain, treadmill training normalizes levels of TRPV1 and TRMP8 cation channels (Yoon et al., 2015), and decreases expression of proinflammatory cytokine receptors IL-1R, IL-6R, and TNFR1 in the DRG . Also, treadmill training after sciatic nerve ligation prevents the increase in chloride transporter NKCC1 in the DRG of rats (Lopez- Alvarez et al., 2015). Finally, cultured DRGs from sedentary male and female mice with experimental autoimmune encephalomyelitis (EAE) demonstrate increased amplitude of calcium response to KCl stimulation (Mifflin et al., 2019). In animals with EAE with access to voluntary running wheels, the calcium response to KCl stimulation is decreased but in females only (Mifflin et al., 2019).
Lastly, repeated exercise modulates levels of pro-inflammatory cytokines in the DRG. Treadmill training reduces DRG levels of IL-1β, IL-6, and TFNα in rats with diabetic induced neuropathic pain Yoon et al., 2015). Also, treadmill training reduces DRG levels of IL-1β and IL-6 following induction of post-incisional pain in rats . Thus, exercise modulates immune function at the level of the DRG through alterations in presence of immune cells, transcription, neurotrophic factors, excitatory neurotransmitters, ion channels and pro-inflammatory cytokines.

Systemic changes
Induction of pain models can produce systemic alterations in immune system function including circulating chemokines and cytokines which is modulated by routine exercise (Fig. 1). Following sciatic CCI in rats, cultured PBMCs from blood stimulated with LPS produce higher amounts of IL-1β and IL-10 when compared with pain free animals (Grace et al., 2016). Voluntary wheel running prior to CCI injury prevents the increased release of IL-1β and IL-10 from LPS stimulated PBMCs (Grace et al., 2016). In a mouse model of EAE, cultured immune cells from the spleen increased secretion of the pro-inflammatory cytokines of IFNγ, TNFα, and IL-17a when stimulated with myelin oligodendrocyte glycoprotein (MOG 35-55 ); voluntary wheel running prevents the MOG 35-55 stimulated increase of TNFα, in females only (Mifflin et al., 2019).
Sciatic nerve CCI results in increased circulating levels of the chemokines CCL2, CCL3, and CXCL1 in rats (Grace et al., 2016). Voluntary wheel running prior to CCI, attenuates circulating levels of these chemokines and elevates levels of the anti-inflammatory cytokine IL-10 ( Grace et al., 2016). Similarly, prior voluntary wheel running, prevents the systemic elevation of the chemokines CXCL1, CCL5, and CXCL5 in response to induction of endometriosis in rats (Appleyard et al., 2021). In mice, treadmill training prior to induction of gout, decreases the expression of TLR2 on neutrophils and levels of CXLC1 in circulation (Jablonski et al., 2020). Lastly, voluntary wheel running reduces circulating levels of IL-1β and IL-6 mRNA in mice with high-fat diet induced pain (Cooper et al., 2017). Thus, repeated exercise shows its ability to produce systemic immune effects suggesting its beneficial effects are not limited to the group of muscles subject to training.

Human studies
In healthy human subjects, acute bouts of exercise transiently increase inflammation, while regular exercise may act as an antiinflammatory treatment (Walsh et al., 2011, Campbell and Turner, 2018, Klasson et al., 2022, Sellami et al., 2021, Zhao et al., 2012, Chastin et al., 2021, Shimizu et al., 2008, Simpson et al., 2020, Ferreira et al., 2018, Cantó et al., 2018, Scheffer and Latini, 2020, Nieman and Pence, 2020, Nieman and Wentz, 2019, Suzuki, 2018, Cerqueira et al., 2019. Acute bouts of exercise elicit a cascade of inflammatory events measured by changes in PBMC counts, lymphocyte subpopulations (T cells, B cells, natural killer (NK) cells), granulocyte, neutrophil and NK cell activity, lymphocyte proliferation, and cytokine levels in plasma (Nieman, 1997, Gabriel and Kindermann, 1997, Pedersen, 1991, Gonçalves et al., 2019, Moldoveanu et al., 2001. Some suggest acute bouts of exercise may transiently redistribute immune cells to peripheral tissues, leading to immune system adaptations and improved immunocompetence (Campbell and Turner, 2018;Simpson et al., 2015;Simpson et al., 2020). On the contrary, repeated bouts of physical exercise may enhance the immune response, reinforce antioxidative capacity, reduce oxidative stress, and increase the efficiency of energy generation, thus inducing an anti-inflammatory state and reducing the incidence of inflammatory diseases (Laddu et al., 2021, Scheffer and Latini, 2020, Pedersen and Hoffman-Goetz, 2000. Multiple factors may influence the inflammatory response following exercise, including exercise type, intensity, duration, and clinical conditions including individuals with chronic pain (Nieman et al., 2012, Moldoveanu et al., 2001, Allen et al., 2015, Bigley and Simpson, 2015, Silveira et al., 2016, Hennigar et al., 2017, Peake et al., 2008. For example, single bouts of prolonged, high-intensity exercise impair T cell, NK cell, and neutrophil function, alter cytokine balance, and blunt immune responses to primary and recall antigens in vivo in healthy individuals. In contrast, single and routine bouts of low-tomoderate intensity exercise enhance immune function including reduced number of senescent T cells, increased T cell proliferation, lower levels of circulating inflammatory cytokines, and increased neutrophil phagocytic activity , Spielmann et al., 2011, Simpson et al., 2012, Shinkai et al., 1995, Pedersen and Bruunsgaard, 2003, Yan et al., 2001. Therefore, the evidence in healthy humans suggests low-to-moderate intensity exercise may lead to beneficial effects on immune function which in theory should lead to improved pain and treatment outcomes. The beneficial effect of routine low-to-moderate intensity exercise on immune function in healthy humans perhaps mimics the reported animal literature in prior sections which primarily consist of voluntary wheel running or graded increases in exercise exposure. However, the clinical literature is comprised of exercise protocols ranging from low-to-high intensity exercise which are summarized below and in Table 2. Multiple chronic pain conditions are linked to etiology associated with changes in immune system function including fibromyalgia, osteoarthritis, rheumatoid arthritis, peripheral neuropathy, low back pain, and complex regional pain syndrome (Alivernini et al., 2020;Bäckryd et al., 2017;Ernberg et al., 2016;Held et al., 2019;Hysing et al., 2019;Teodorczyk-Injeyan et al., 2018;. While the effects of exercise on immune function have been investigated and described in healthy populations, the evidence in chronic pain populations is less clear due to conflicting evidence, varying exercise protocols, and limited study.   (Andersson et al., 2020, Bjersing et al., 2012, Bote et al., 2013, Bote et al., 2014, Cheng et al., 2015, Chua et al., 2008, Helmark et al., 2010, Hunt et al., 2013, Jablochkova et al., 2019, Oliveira et al., 2017, Ortega et al., 2012, Ortega et al., 2009, Salm et al., 2019, Samut et al., 2015Simão et al., 2012;Wåhlén et al., 2022).

Acute/single bouts of exercise
Despite the relevance to exercise prescription there has been limited investigation into the impact of acute exercise on immune function and pain in clinical populations. Prior study suggests individuals with fibromyalgia who have a proinflammatory state of elevated cytokines IL-1α, IL-6, and IL-8 at baseline experience a defective stress response to high-intensity exercise as measured by reduced circulating growth hormone and increased pain following exercise, suggesting the inflammatory status of patients influences exercise response (Ross et al., 2018). However, the change in circulating cytokines following exercise and the relation to clinical symptoms are unclear. The impact of acute bouts of low-to-moderate intensity exercise on immune function are equivocal. Bote et al. (2013) showed a decrease in IL-8 concentration in circulating serum and stimulated release of IL-1β, TNF-α, and IL-6 by monocytes following a single bout of moderate intensity cycling in fibromyalgia; however, the changes in clinical pain were not evaluated. In individuals with knee osteoarthritis, an acute bout of moderate-intensity resistance exercise increases the anti-inflammatory cytokine IL-10 locally within the synovium and peri-articular area of the knee joint (Helmark et al., 2010); again, clinical pain was not assessed. In contrast, local effects of exercise are not observed in fibromyalgia, as a 20-minute bout of lowintensity exercise did not change pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF) in the exercising muscle (Christidis et al., 2015). A systematic review of 6 articles examining a single bout of exercise showed unclear results for a directional preference circulating cytokines in fibromyalgia (Andrade et al., 2018). Of the available evidence, the relation between changes in circulating cytokines to changes in pain in clinical populations following a single bout of exercise is limited. Further investigation into the immune response following single bouts of exercise in chronic pain is needed to examine the positive and negative effects on the immune system and how these relate to symptoms.

Routine exercise
Since regular exercise has the capacity to modulate immune function to enhance anti-inflammatory activity of immune cells in healthy people and animal models (Walsh et al., 2011, Calcaterra et al., 2022, Klasson et al., 2022, Campbell and Turner, 2018, Zhao et al., 2012, Shimizu et al., 2008, Sellami et al., 2021, Chastin et al., 2021, Oliveira-Fusaro et al., 2020, Gregory et al., 2018, da Silva et al., 2015, Nijs et al., 2014, Polli et al., 2019, Clark et al., 2017, Liu and Wang, 1987, Kohut et al., 2001, Baek et al., 2020, Cannon and Kluger, 1984, it is important to determine if regular exercise modulates the immune system in chronic pain populations. Prior randomized controlled trials using various modes of exercise ranging from aerobic (cardiorespiratory), muscle strengthening, stretching, and aquatic based exercise show varying effects on immune function. Ortega et al (2009) showed 8 months of aquatic exercise reduced monocyte evoked release of proinflammatory markers (IL-1, IL-6, TNFα), circulating C-reactive protein, and increased anti-inflammatory marker (IL-10) in individuals with fibromyalgia. Thus, four months of routine exercise improved the balance of pro-and anti-inflammatory cytokine production and lead to better regulation of monocyte function in individuals with fibromyalgia. There was also improvement in quality of life and function, however direct correlations to immune changes were not investigated. Furthermore, Bote et al (2014) showed 8-months of routine aquatic exercise reduced neutrophil chemotaxis, circulating IL-8 and noradrenaline at 8 months, while there were no changes at 4 months in individuals with fibromyalgia, suggesting longer duration of routine exercise may be necessary to induce changes in immune function that promote anti-inflammatory adaptations. Concomitant improvements in quality of life support the hypothesis that exercise-induced changes in inflammatory function may impact clinical symptoms and function. Despite these promising results, others have found routine exercise did not change various markers of immune function (Table 2) including PBMC subsets in RA, (Baslund et al., 1993;Rall et al., 1996) cytokine gene polymorphisms in knee OA, (Nicklas et al., 2005) and plasma cytokines in migraine (Oliveira-Fusaro et al., 2020), muscle in RA (Andonian et al., 2018) and muscle in fibromyalgia (Ernberg et al., 2018). Variation in the prior literature can be attributed to multiple factors including focusing systemic vs local effects, small sample sizes, and different pain conditions. On the other hand, in women with trapezius myalgia, there is an increased numbers of macrophages in muscle which could be related to the regenerative myogenic response to strength training (Mackey et al., 2011).
In addition to factors such as exercise type, intensity, duration, and tissue type which may impact outcomes, the mixed results from the available literature may be attributable to low sample size in the majority of studies, use of mixed populations of immune cells, use of varying methods to investigate immune function, and use of different stimuli to evoke cytokine release from immune cells. In addition, the potential influence of biological sex on changes in immune function following exercise in humans is unknown as most studies have included females only or a limited number of males in their sample. Despite these limitations, preliminary studies show that exercise can alter systemic cytokines and reduce systemic inflammation, a proposed mechanism of chronic pain in humans. Additionally, there has been a lack of investigation into the relation of changes in clinical pain measures to immune system changes.

Animal research
The animal research demonstrates that regular exercise can modulate the immune system response to induction of pain at the site of injury, in the DRG, and in the systemic circulation. This suggests that repeated exercise bouts produce whole body effects, and its benefits are not restricted to the location of training. Also, several types of exercise programs are beneficial at preventing or reducing pain suggesting patient preference should be heavily considered when prescribing exercise programs to help increase adherence. Lastly, routine exercise shows positive benefits when performed either before or after onset of pain. Therefore, exercise programs should still be followed in the absent of pain to help prevent pain exacerbations. While this review highlighted several articles showing regular exercise-induced changes in the immune response, many questions remain regarding the underlying mechanisms through which exercise alters immune function. Future research should focus on upstream and downstream mechanisms for how exercise modulates immune phenotype and alters cytokine release. Understanding these mechanisms could identify novel therapeutic targets and assist with exercise prescription for the treatment of chronic pain. Also worth noting is the lack of research utilizing both sexes to study the effectiveness and mechanisms of exercise-induced analgesia. This is especially concerning in studies exploring the immune system since there are significant sex differences in immune mechanisms in the generation of pain (Baskozos et al., 2019;Cowie et al., 2019;Mecklenburg et al., 2022;Mogil, 2020;Plumb et al., 2023;Sorge et al., 2015). Of the papers that did include both males and females, sex differences were found in exercise immune mechanisms (Mifflin et al., 2019;Lesnak et al., 2022) and are highlighted above. Therefore, future work needs to include both male and female animals to further elucidate sex differences in exercises ability to modulate the immune system to prevent and alleviate pain.

Human research
Acute bouts of exercise and routine exercise training influence the immune response in individuals with chronic pain. The ability of the immune system to adapt towards an anti-inflammatory state with routine exercise intervention may provide promising outcomes for the treatment of chronic pain. In addition to pain, the anti-inflammatory effects of routine exercise may provide multiple health benefits including reduced cardiovascular disease, obesity, type 2 diabetes, risk of cancer, sarcopenia, and dementia. The available evidence does not support specific exercise guidelines that promote an anti-inflammatory state as multiple factors may influence the inflammatory response to acute and chronic exercise. Appraisal of the available literature suggests it may be particularly important to adjust the characteristics of and exercise programs in order to obtain an anti-inflammatory response and/or restore an optimal anti-and pro-inflammatory state. When managing patients with chronic pain, clinicians should consider initiating at low to moderate intensity exercise that is adapted to the individual patient to help lessen the pro-inflammatory response that is typically seen following acute bouts of high-intensity exercise. This is of particular importance when prescribing exercise to untrained individuals or those with limited physical capacity. An exercise program should be tailored to the individual needs and ability of the patient to help reduce barriers to routine physical activity and promote compliance to long-term intervention. The treatment goal should be to establish a long-term exercise program which improves pain, activities of daily living, quality of life, and immune function. In addition to exercise effects, a comprehensive treatment plan may lead to additive or synergistic effects on immune function with targeted pharmacological agents and dietary modification or supplementation. Future research should capitalize on advances in assessment of immune system and exercise immunology in relation to clinical and behavioral assessments in humans and animals. Further research investigating the link between the immune system and acute and chronic exercise in chronic pain conditions is needed to identify unique insights into potential mechanisms underlying the pathology of pain, therapeutic targets, and management strategies.

Conclusion
The available animal and human literature provide strong indication that routine exercise leads to adaptations in immune function which may relate to pain behaviors in animals and self-reported symptoms in humans. While single bouts of exercise may lead to transient increases in inflammation, routine exercise may be beneficial as an intervention or preventative treatment to promote an anti-inflammatory immune profile which can subsequently reduce pain. Thus, repeated bouts of exercise should be utilized as a first-line intervention for individuals with chronic pain due to its ability to positively impact various physiological systems to prevent and alleviate pain.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.