Is chronic pain as an autoimmune disease?

ABSTRACT Autoimmune diseases frequently occur in females, and a parallel sexually dimorphic suffering is observed in individuals who suffer chronic pain. Though perception and environment influence the chronicity of pain, this review illustrates examples of specific, evolutionarily preserved, physiological parameters that may be responsible and differentially contribute to chronic pain and affect treatment outcomes in females and males. In females, the immune system may be continuously “primed,” potentially due to the presence of two X chromosomes, each bearing a number of genes involved in immune responsiveness. In the event of nerve injury, declining parity rates could be having repercussions via increased rates of chronic pain or less effectiveness to therapies, which may be associated with a heightened immune cell infiltration into damage-associated sites. Additionally, the female hormone estradiol is both neuroprotective and neurodegenerative, with reproductive cycle– and age-dependent outcomes. There is therefore a need to study neuro-immune-endocrine crosstalk in the context of chronic pain. Autoantibodies have been associated to neural antigens with sensory pathway hyperexcitability in patients, and self-antigens need to be identified by damaged nerves remain to be discovered. Specific T cells release pronociceptive cytokines that directly influence neural firing, and T lymphocytes reactivated by specific antigens may elicit neuroprotective effects by secreting factors that support nerve repair. Modulating immune cells could therefore be a mechanism by which nerve recovery is promoted, with sex-specific outcomes. Investigating neuroimmune homeostasis may inform the selection of specific treatment regimens for females or males and hence may improve chronic pain management by recalibrating the influence of the immune system on the nervous system.


Introduction
Why are females more likely to experience chronic pain than males? Given that pain is complex, involving biological, psychological, and social factors, this question remains difficult to answer, yet an answer is critical to improve upon current pain management practices. Addressing sex differences in the clinic will be a turning point in designing and developing optimized therapeutics and improving the efficacy of existing analgesic agents for treatment needs of males and females based on biological differences.
The premise that pain is less common in males than in females is supported by significant evidence (for reviews, see Fillingim et al., 1 Bartley and Fillingim,2 and Pieretti et al. 3 ). Back pain, migraine, and osteoarthritis are reported to be less common in males than in females. 4 A questionnaire-based investigation of over 45,000 individuals in 16 countries in Europe examined the incidence of chronic pain in males and females over their life spans. Though the incidence of pain increased in both males and females as they aged, the number of males and females reporting chronic pain incrementally diverged. This divergence commenced in early adulthood, with the gap between the sexes widening with age. 5 It should be noted that sex differences do not apply to all painful chronic conditions. For example, though pain associated with autoimmune conditions affects more females than males, this is not the case for disorders such as diabetes, in which neuropathy occurs at equal rates in both sexes. 6 In addition, the prevalence of chronic pain in males and females may change over the course of their lives, in a manner dependent on the pain condition. As an example, the incidence of fibromyalgia increases with age, as does its higher prevalence in females. In comparison, the occurrence of migraineassociated pain is highest in females between the ages of 20 and 30, declining after menopause. 7 Females also reported consistently greater sensitivity to temperature and muscle-associated pain than males. 4 Of note, the magnitude of these sex differences varies depending on the survey questions posed to an individual, as well as the methods employed to measure pain. The latter may include an assessment of the pain threshold (the point at which a stimulus such as temperature or pressure is first observe as painful), tolerance (the increased amount of pain that an individual can tolerate), and unpleasantness (how unpleasant a given stimulus is perceived to be).
The reasons for acquiring a mechanistic understanding of how sex influences pain have largely focused on nociceptors and neuronal signaling. Usually, nociceptors detect potentially harmful stimuli, including, for example, heat, noxious chemicals, or changes in pressure. In response to nerve injury, these neurons are aberrantly activated and can occur in the absence of such stimuli, with the possibility for a nonnoxious stimulus, such as gentle stroking of the skin, causing severe pain. Hyperexcitability of nociceptors, via N-methyl-D-aspartate receptor activation, eventually leads to the chronification of pain. 8 Distinct hormone profiles between males and females may influence sex differences in hyperexcitability. For instance, glutamate currents in the dorsal root ganglia (DRG) were observed to be higher in female compared to male rats, possibly via a mechanism involving 17-β-estradiol. 9 Also certain genes involved in ion transport, may be involved in producing a hyperexcitable response, are upregulated in female rats. 10 In the field of pain research, the DRG have been of considerable interest, because these nodular enlargements of nerve bundles contain the somata of peripheral nociceptors. Electrical impulses produced by nociceptors are transmitted as action potentials along the axons to the DRG, with signals further relayed into the spinal cord. The ascending pain pathway continues with the spinal cord transmitting peripheral input to the brain, which processes the input, "interpreting" it as painful and initiating descending signals.
Interestingly, a sex difference was detected in some peripheral immune cells that had infiltrated into the DRG. 11 In both males and females, a greater number of immune cells such as macrophages, monocytes, and neutrophils were present in the DRG following a paininducing nerve injury in mice, and more B and T lymphocytes were observed in males and females, respectively. 11 With regard to common sex differences in the immune system, in a healthy population, a significantly lower number of peripheral T lymphocytes was recorded in men compared to women, 12 with evidence supporting an increased adaptive immune response in women. 13,14 Importantly, the approach carried out by Lopes et al. 11 indicates that sex differences in pain sensitivity may not stem from transcriptional changes in the nociceptors themselves and that (an)other process(es), likely associated with the peripheral immune system, may underlie differences in pain chronification between males and females.
The message, across disciplines, is that it has become critical to gain a deeper understanding of the basis for sex differences. 15 As pain research advances to account for the analgesic needs of males and females, the sex chromosomal status and fluctuating concentrations of gonadal hormones will need to be consistently addressed. This will require assessments of their complex, and likely interconnected, effects on immune responses and the vasculature, which in turn influence the activity of the central and peripheral nervous system. Various hypotheses have been suggested that could explain the physiological mechanisms that contribute to sex differences in pain signaling. This review first provides an overview of chronic pain with an emphasis on possible links with autoimmune activity.

Immune Responses after Nerve Injury
Immune activity that occurs in response to nerve damage contributes to neuropathic pain. Rats with a nerve cuff on the sciatic nerve experienced both mechanical allodynia and thermal pain hypersensitivity in the injury-associated limb. 16 Macrophages, neutrophils, dendritic cells, and T lymphocytes infiltrated the partially ligated sciatic nerve and the corresponding ipsilateral DRG, whereas B-lymphocyte infiltration appeared to be primarily limited to the site of injury. 16 Macrophages and T helper (Th) lymphocytes secrete pro-inflammatory cytokines, such as interleukins and tumor necrosis factor alpha (TNF-α). 17 TNF-α can activate nociceptive nerve terminals, leading to nociceptor sensitization and neuropathic pain, 17,18 and neutrophil infiltration into the region surrounding the injured sciatic nerve has also been shown to contribute to nociception in mice. 19 Furthermore, a significant increase in the activation of microglia and astrocytes occurred in the ipsilateral spinal dorsal horn in these injured animals. 16 Interleukin 17 (IL-17), which is produced by Th17 lymphocytes, may be involved in triggering this response. 19 IL-17 knockout in mice with a partial sciatic nerve ligation led to reduced mechanical nociceptive responses, attenuation of macrophage and T-cell infiltration into the damaged region and DRG, and reduced microglial and astrocytic activation in the spinal dorsal and ventral horns. 20 Furthermore, neutrophil infiltration at the nerve (sciatic nerve) was detected following administration of IL-17 into this region. 20 Interestingly, IL-17, a cytokine, has been associated with and linked to inflammatory autoimmune pathologies such as rheumatoid arthritis and multiple sclerosis, 21,22 and this cytokine was recently postulated as a potential biomarker in patients with sciatic nerve damage. 23 In addition, compared to an autopsy control group with nondegenerative tissue, IL-17 was significantly higher in patients with degenerative disc disease and herniated intervertebral disc disease, 24 both of which can lead to impingement of the sciatic nerve.
Interferon gamma (IFN-γ) is produced by Th1 cells and stimulates the expression of major histocompatibility complex class II, which promotes activity in macrophages and induces the production of other cytokines. 25 Furthermore, IL-10, produced by several immune cell types including B cells and Th2 lymphocytes, mediates macrophage activity at the location of sciatic nerve injury, attenuating pro-inflammatory cytokine production, promoting proregenerative activity in macrophages, and contributing to axon regeneration. 26 Although it promotes antibody production, it also has regenerative activities via IL-10 and has been implicated in several painful autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. 27 Indeed, IL-10 contributes to the production of autoantibodies in systemic lupus erythematosus. 28 Several studies have demonstrated a role for both IFN-γ and IL-10 in the immune response to sciatic nerve damage. A measurable change in the expression of IFN-γ, IL-10, and the IFN-γ/IL-10 ratio has been recorded in rats following a sciatic nerve injury. 29 Interestingly, IL-10 was detected as another potential biomarker in human patients with sciatica, 23 and levels of IFN-γ were significantly higher in patients with herniated intervertebral disc disease. 24 Andrade et al. 30 consistently detected IL-10, TNF-α, and IL-1β in the cerebrospinal fluid of individual patients with thoracic disc herniation. 30 IL-10 and TNF-α were responsive with opposite effects to high pain scores. 30 Furthermore, increased levels of IL-1β and IL-6 have been reported in patients with sciatic pain due to lumbar disc herniation. 31 Though these cytokines exhibited no significant association with pain scores in these patients, IL-6 has been implicated in several autoimmune pathologies such as rheumatoid arthritis. 32 TNF-α and IL-1β have been examined as potential biomarkers in patients with sciatica, with TNF-α exhibiting a moderate association with pain. 23 Though the application of TNF-α-based therapies aimed at treating sciatica achieved mixed levels of success, attenuating TNF-α activity using infliximab significantly reduced pain in a small group of patients with disc herniation-induced sciatica when compared to treatment with saline, 33 although sex differences were not evaluated. It is noteworthy that, though TNF-α and IL-1β contribute to neuropathic pain in mice with a nerve injury, these cytokines are required for functional recovery of the nerve. 19 Indeed, recovery was impaired in TNF, IL-1β, and TNF/IL-1β knockout mice. 19 Collectively, these findings indicate that dysregulation of pro-inflammatory cytokines may contribute to neuropathic pain that occurs in response to nerve injury.

Microglia
In a normal physiological state, microglia are "resting" yet dynamic, continuously scanning their environment for any changes that could alter homeostasis. Upon exposure to certain signals, microglia are activated to perform innate immune functions. 34 In this later state, microglia drive synaptic alterations within the dorsal horn of the spinal cord, representing a key pronociceptive event. 35 It is thought that microglia that fail to return to a resting state contribute to persistent neuropathic pain. 36,37 The finding that the purinergic receptor 4, a highly sensitive ligand-gated ion channel, is expressed by microglia highlighted their importance in mediating peripherally induced nociceptive hypersensitivity in rodent models of pain. 38

Autoantibodies
Autoimmunity represents a pathological process whereby antibodies target self-antigens and, in certain contexts, contribute to neuropathic pain. In many cases, autoimmune disorders involve B cell-produced immunoglobin G (IgG) antibodies directed against specific antigens. In a recent study, Cuhadar et al. 37 identified peripheral A and C fiber-mediated nociceptor sensitization as a major mechanism by which autoantibodies may produce pain in complex regional pain syndrome. 37 The authors subjected female mice to a minor experimental insult concomitant with the administration of IgG isolated from patients with chronic complex regional pain syndrome, which resulted in persisting mechanical and thermal sensory changes. Furthermore, the degree of transferred hyperalgesia correlated with the dose of IgG and donor patient pain scores; reduced IgG-mediated nociceptive responses were recorded in animals receiving IgG transferred from patients reporting moderate levels of pain. 37 Importantly, in ex vivo cutaneous nerve preparations, the spontaneous and evoked action potential discharge rates were increased, demonstrating that patient IgG autoantibodies were able to generate nociceptor hyperexcitability. 37

X and Y Chromosomes
If the neurons associated with nociceptive responses turn out not to be the fundamental source accounting for sexual dimorphisms in unresolved pain states and major sex differences associated with human pathologies cannot solely be explained by environmental factors or reproductive hormones, another difference between males and females is responsible for the chronic nature of pain. X and Y chromosome-associated genes exhibit differential expression profiles. This arises independent of an individual's hormonal status and could therefore represent a major driver for the differences between females and males in pathological conditions, including chronic pain. Identification and characterization of genes on the X and Y chromosomes are being actively investigated. The X chromosome represents approximately 2.5% in men (XY) of the total DNA within each cell, referred to as the dosage, which is doubled in women (XX).

The X Chromosome
One of the two X chromosomes has been observed to randomly undergo permanent somatic cell X-inactivation during embryogenesis. The purpose of this process is suggestive that females resemble males in maintaining only one functional copy of the X chromosome per somatic cell. X chromosome silencing occurs via two potential mechanisms, namely (1) epigenetic changes that include chromatin modification 39 and (2) the coating of one X chromosome by the X-inactive specific transcript (XIST). [40][41][42] The second mechanism requires the Ying Yang 1 protein, which activates XIST 43 and attaches it to the X chromosome. 44 It is noteworthy that 10% to 15% of genes that are localized on the X chromosome escape X-inactivation. This results in their biallelic expression and skewed transcript levels in females. 45 X-inactivation silencing occurs primarily to genes within the pseudoautosomal region at the end of the short arm of the X chromosome, denoting nonrecombining sequences. A large number of these genes are associated with a pathological state such as major psychiatric disorder, systemic lupus erythematosus, Rett syndrome, and thyroid autoimmunity. [46][47][48] Many sexually dimorphic variances in disease susceptibility could potentially be attributed to altered gene expression that is associated with an escape from X chromosome inactivation. To support this concept, sex chromosome has been linked to abnormalities of brain disorders, 49 such as X chromosome inactivation and its association with neural development, disease, 50 and murine Ying Yang 1 expression, which plays a role in both morphine analgesia and inflammatory pain. 51

The X Chromosome and Pain
A number of reports support that the X chromosome is associated with higher rates of pain 2,4,52,53 and posttraumatic stress as observed in females compared to males. 54,55 Sex differences have also been described in animal models, with increased hyperalgesia 56,57 and insufficient fear extinction reported in females. 58,59 Investigations have shown that a large number of individuals who develop chronic musculoskeletal pain 60,61 and/or symptoms of posttraumatic stress 62 after an automobile accident are females. These females who have XIST are found to have dysregulated chronic pain. -48 An investigative study by Yu et al. 63 observed that, during the initial stages following a car accident, 40 genes originating from the X chromosome were differentially expressed in females and were responsible for the development later of chronic musculoskeletal pain and/or signs of posttraumatic stress compared to those who recuperated. 63 By comparison, 25 X chromosome genes were differentially expressed males, with the authors noting that this repertoire was different from those identified in the set from females. In males, two well-defined clusters were identified by pathway analysis and were enhanced for genes previously shown to not be responsible for X chromosome inactivation. These group of genes were based on upregulated gene expression associated with the eukaryotic initiation factor 2 signaling pathway or IL-2 pathway. 63

Y Chromosome
All Y-linked genes are expressed, unlike X chromosome genes, from genes that are duplicated and hemizygous, except in cases of aneuploidy. Reconstruction across mammalian species based on evolution suggests that preservation of specific parts of the Y chromosome occurs randomly over time. 64 It appears that the gene content of the Y chromosome has selectively become specialized to maintain the ancestral dosage of homologous XY gene pairs that act as key regulators of transcription, translation, and protein stability in a number of tissues. Several ancestral genes on the human Y chromosome seem to have survived, with four genes (HSFY, RBMY, SRY, and TSPY). These encoding isoforms have functionally diverged from their X-encoded homologs (HSFX, RBMX, SOX3, and TSPX) to ensure male reproductive development or gametogenesis. 64 It appears that in mammals, the SRY gene is the main driver of male development. Several ubiquitously expressed ancestral genes exhibit subtle functional differences from their X-linked homologs. Eight of the regulators of transcriptional activity present in a number of human tissues, including DDX3X/Y, EIF1AX/Y, KDM5C/D, RPS4X/Y, TBL1X/Y, USP9X/Y, UTX/Y, and ZFX/Y, illustrate a biochemical sexual dimorphism that is directly linked to genetic differences between the X and Y chromosomes. Several of these genes (EIF1A, UTX) have been associated with chronic pain, as suggested earlier. Thus, the Y chromosome could have an underappreciated role in broader sex differences that influence processes other than testis determination and spermatogenesis that impact normal physiology as well as pathology.
Current investigations are examining the possibility that the male pattern of neural development may be a direct consequence of Y chromosome-related gene expression or an alternate indirect result of the Y chromosome's relation to testosterone production. 65 Y-linked genes in chronic pain conditions will be of great interest in the future.

XY Chromosomes and Immune Cells
The X chromosome is responsible for an immune response because it harbors a number of genes involved in the immune process, with women having a higher dosage of these genes. 66,67 Furthermore, activation of normally inactive portions of the X chromosome could potentially result in the breakdown of immune tolerance in females. 66 In a recent investigation, the transcriptome of 11 immune cell types (B1A and B2 cells, CD4 + and CD8 + T cells, dendritic cells, gamma delta T cells, granulocytes, macrophages, natural killer cells, natural killer T cells, and Treg cells) was profiled in 92 female and 91 male mice. 68 Both Xist and Eif2s3y were differentially expressed in males and females in 9 of these cell types. There was higher male expression of autosomal Rps17 (40S ribosomal protein S17) in Treg cells; Forty one other autosomal cell type-specific genes were differentially expressed between males and females and were limited to macrophages. 68 Approximately 26 of these genes were highly expressed in female macrophages, including genes involved in the complement system, namely, Fcgr2b (encoding FcγRIIB; inhibitory) and Fcgr3a (encoding FcγRIIIA; activating).

Pregnancy Compensation Hypothesis
Investigators have postulated that the female immune system is prepared for the presence of a placenta, even in its absence. Recently it has been postulated that the need for females to compensate for the robust immune system activity that occurs during pregnancy has been ancestrally manifested. 69 Differences in inherited X and Y chromosome gene dosage and gene content have resulted in the specialized immune activity of pregnant females to enhance survival in the presence of a placenta in an immunologically challenging environment, with female hormones directly impacting this process. Conceptually this could help to understand the sex differences in immune function and its implications with associated pathologies.
The primary objective of the pregnancy compensation hypothesis is that all placental mammals have evolved to support high parity across a life span. 69 There are risks associated with pregnancy, and female physiology has evolved to adapt with key immune system developments. The hypothesis is that this evolutionary process was necessary to counter the challenges of the placenta, which enables the maternal immune system to alter its normal activity, thus ensuring that the developing fetus is not rejected as "foreign." 69 The placenta could potentially be detected as a foreign organ, because the chorion frondosum develops from a blastocyst. Hence, the placenta and fetus are considered as sites of immune privilege, with both processes resulting in maternal tolerance. An example of this mechanism is placental secretion of phosphocholinated neurokinin B. 70 This mechanism is thought to provide a "cloaking" system to the placenta, given that phosphocholines are frequently used by parasitic nematodes to evade host detection. 71 Moreover, fetal small lymphocytic suppressor cells are able to inhibit maternal cytotoxic T lymphocytes by blocking their response to IL-2. 72 Nevertheless, dampening specific immune responses has the potential for increased adverse effects, making females sensitive to pathogens with a potential adverse effect on the developing fetus. To compensate, a female's immune system is primed throughout adulthood to constantly scan for pathogens, even during immune quiescence accompanying pregnancy. 69 Thus, it is plausible that the female immune system is overprimed without purpose and, consequently, aberrant responses occur, initiating the development of autoimmune diseases. 69

VGLL3 and BAFF
The pregnancy compensation hypothesis explains why the incidence of autoimmunity is higher in females than in males. In addition, it has recently been shown that females express higher epidermal levels of a putative transcriptional co-factor, vestigial-like family member 3 (VGLL3), independent of biological age and gonadal hormone regulation. 73 VGLL3 exhibits female-specific nuclear localization, which suggests that it is implicated to play a key role in sexually dimorphic transcriptional regulation of its target genes. Knockdown of VGLL3 in vitro results in decreased expression of select femalebiased immune transcripts, including BAFF (B cell-activating factor, also known as B-lymphocyte stimulator and TNFSF13B). This is the target of belimumab, the only biologic treatment that is currently used to treat systemic lupus erythematosus. 74 Similarly, males with this autoimmune condition demonstrate upregulated expression and nuclear localization of VGLL3 in their inflamed epidermis. 73 Interestingly, skin-directed overexpression of Vgll3 in female mice causes systemic autoimmunity, with symptoms similar to those observed in patients with systemic lupus erythematosus. 75 B-cell expansion, autoantibody production, and immune complex deposition are shown to ultimately contribute to tissue damage. They are all found to be engaged, with upregulated BAFF occurring as a consequence of overexpressed Vgll3 in females, further demonstrating it as a driver of sex-specific autoimmunity. 75 Though specific gene products such as VGLL3 and BAFF may provide context to the sexually dimorphic mechanisms underlying differential engagement of the immune system in males and females, the pregnancy compensation hypothesis explains why this difference fundamentally occurs. Pregnancy may have significantly shaped the evolution of the immune system, providing selective pressure to engage more X chromosome-associated genes for optimal regulation of relevant immune responses. It will be of considerable future interest to identify the specific components of the immune system that respond to the placenta, which may provide novel targets to develop female-specific treatments for conditions with an autoimmune component, including chronic pain.

Sex Hormones-Select Examples
Although X and Y chromosomes may shape male and female responses to pain, gonadal hormones are equally important in this process. It has been shown that in newborn mice, sex chromosome complement (XX vs XY) together with gonadal sex influences the development of nociception, as well as responses to analgesic drugs. 76 Compared to the effects of estrogens, relatively little is known regarding how the neuroimmune system is modulated by androgens. It is now generally accepted that testosterone is immunosuppressive, with clinical evidence supporting that this gonadal hormone may protect against autoimmune disease. 77,78 In males, androgen deficiency stemming from hypogonadotropic hypogonadism and Klinefelter's syndrome (XXY) is associated with an increased risk of autoimmune disease. For example, in patients with Klinefelter's, a staggering 18-fold increase in the incidence of systemic lupus erythematosus has been reported, with clinical remission occurring in response to androgen therapy. 79 Testosterone deficiency also increases autoimmune disease-modeled activity in orchiectomized mice, 80,81 and androgen therapy improved male survival in a mouse model of systemic lupus erythematosus. 82

Conclusions
Sex differences have a biological basis and are undoubtedly complex. Sex-associated steroids cause activation of some pain responses and analgesic efficacy in adults. Perinatal dimorphisms in testosterone levels produce enduring organizational differences in males and females. Various lines of evidence support that immunetriggered conditions exhibit a sex bias in children prior to the onset of puberty, 83,84 and therefore it is important to explore facets other than gonadal hormones to understand sex differences in chronic pain. In addition to the action of sex hormones on nociceptive circuits, genes mapping to the X and Y chromosomes are considered to be important players, with the neuroendocrine system modulating the effects stemming from the sex chromosome complement. Hence, research in chronic pain, from a patient perspective, will require fundamentally improving therapeutic interventions to provide precision medicine for feamales and males. It potentially may require the direct manipulation of gonadal hormones in relevant female and male animal models at different life stages. This will be important to determine how hormonal and sex chromosomal influences interact to modulate the neuroimmune system. It will provide a better fundamental understanding of their roles in the development, maturation, and dysregulation of nociceptive circuits.
This review highlights some similarities between chronic pain and autoimmune conditions. Given that the incidence of autoimmune diseases is generally higher in females, it is worth considering that chronic pain could be an autoimmune disease because of their similarities. The body does not operate in isolated systems. It will therefore be beneficial to add momentum to the paradigm shift that is already underway, moving the primary focus from neural firing and central signal processing to peripheral changes that occur in response to nerve damage, including alternations that take place in immune tissues, the lymphatic system, and the vasculature. Novel insights will be gained by considering how perturbations in homeostasis as a whole lead to chronic pain, which may involve very different evolutionarily preserved biological processes in men and women. Thus, considering chronic pain as an "autoimmune disease" could potentially open up new avenues for clinically relevant treatment options. This concept will increase the scope and potential for novel chronic pain therapeutics.

Disclosure Statement
The author has no conflict of interest to report.

Funding
This work was supported by the Michael G. DeGroote Institute for Pain Research and Care Seed Funding, McMaster University with seed funding.