Mast Cells, Neuroinflammation and Pain in Fibromyalgia Syndrome

Fibromyalgia Syndrome (FMS) is a disorder of chronic, generalized muscular pain, accompanied by sleep disturbances, fatigue and cognitive dysfunction. There is no definitive pathogenesis except for altered central pain pathways. We previously reported increased serum levels of the neuropeptides substance P (SP) and its structural analogue hemokinin-1 (HK-1) together with the pro-inflammatory cytokines IL-6 and TNF in FMS patients as compared to sedentary controls. We hypothesize that thalamic mast cells contribute to inflammation and pain, by releasing neuro-sensitizing molecules that include histamine, IL-1β, IL-6 and TNF, as well as calcitonin-gene related peptide (CGRP), HK-1 and SP. These molecules could either stimulate thalamic nociceptive neurons directly, or via stimulation of microglia in the diencephalon. As a result, inhibiting mast cell stimulation could be used as a novel approach for reducing pain and the symptoms of FMS.


INTRODUCTION
Fibromyalgia Syndrome is a disorder of chronic generalized muscular pain, stiffness, generalized fatigue, sleep abnormalities, Schmidt-Wilcke and Clauw, 2011;Clauw, 2014) and cognitive problems (Theoharides et al., 2015b;Hauser et al., 2019) assessed by the FSQ (Ferrari and Russell, 2013), which has about 93% sensitivity and 92% specificity (Clauw, 2014). FMS affects about 5% of adults, primarily women 20-60 years of age (Branco et al., 2010) and belongs to a family of overlapping painful conditions (Table 1) known as CSS (Yunus, 2007;Theoharides, 2013). Central sensitization is recognized as the main mechanism involved (Woodman, 2013) and is characterized by allodynia, pain from an otherwise non-painful stimulus, (Russell and Larson, 2009) and hyperalgesia (Staud et al., 2001) due to an exaggerated response to a painful stimulus Fibromyalgia syndrome worsen by stress, (Geenen et al., 2002) which augments pain responses (Bote et al., 2012(Bote et al., , 2013. Plasma concentrations of cortisol are increased in the evening, suggesting disruption of the circadian rhythm (Crofford et al., 2004). Serum levels of CRH, which is secreted under stress, were increased in patients with FMS (Tsilioni et al., 2016). CRH was also increased in the CSF of such patients and correlated with severity of pain (McLean et al., 2006). Physiological stress was reported to be the most common trigger in patients with systemic mastocytosis (SM) (Jennings et al., 2014) who also commonly experience FMS (Theoharides et al., 2015d(Theoharides et al., , 2019. We reported increased levels of CRH in the serum of one patient with indolent systemic mastocytosis . CRH can trigger human mast cells to release VEGF without histamine or tryptase (Cao et al., 2005). CRH also has synergistic action with NT stimulating VEGF release. As a result, there is increased vascular permeability in the skin and the blood-brain barrier (BBB) (Esposito et al., 2002;Donelan et al., 2006;Theoharides and Konstantinidou, 2007). Stress also disrupts the gut-blood barrier (Theoharides et al., 1999;Wallon et al., 2008) allowing for gut microbiomeassociated molecules, such as propionate (Minerbi et al., 2019) to enter the brain and contribute to focal inflammation. These results have led to the conclusion that mast cells may serve as "immune gate to the brain" (Theoharides, 1990;Ribatti, 2015).
Levels of the neuropeptide SP (Russell, 1998) and NGF (Giovengo et al., 1999) are elevated in the CSF of FMS patients. NGF has been reported to increase nociception and hyperalgesia (Maren, 2017). The SP receptor NK-1 has been involved in the pathophysiology of pain (Greenwood- Van et al., 2014). We reported increased serum levels of SP, its structural analogue Hemokinin-1 (HK-1) and TNF in patients with FMS (Tsilioni et al., 2016). SP (Theoharides et al., 2010a,b) and NGF (Levi-Montalcini, 1987) can stimulate mast cells. Moreover, SP induced mast cell expression of CRHR-1 (Scholzen et al., 2001). Cerebrovascular mast cells were stimulated by CGRP, (Reynier-Rebuffel et al., 1994;Ottosson and Edvinsson, 1997) which is now well established to participate in the pathophysiology of headaches (Edvinsson, 2018). In addition to neuropeptides, sex hormones can also affect mast cell reactivity. For instance, estradiol augments immune (Kovats, 2015) and allergic (Hox et al., 2015) processes. In particular, we had reported expression of estrogen receptors on rodent mast cells (Pang et al., 1995). We also reported that 17β-estradiol further increased stimulation of mast cells by SP (Theoharides et al., 1993). Such findings may help explain why FMS is more common in women.

Thalamic Mast Cells Secrete Neurosensitizing Mediators
Increasing evidence supports the involvement of mast cells in FMS (Lucas et al., 2006;Pollack, 2014) and comorbid disorders (Theoharides, 2013) as well as other inflammatory Theoharides et al., 2010a) and painful conditions, (Heron and Dubayle, 2013;Chatterjea and Martinov, 2014) as well as neuroimmune interactions (Skaper et al., 2017) (Figure 1). Chronic urticaria, which involves stimulation of skin mast cells is more common in FMS (Torresani et al., 2009). Moreover, mast cells are significantly increased in the papillary dermis of FMS patients (Blanco et al., 2010). The chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2) and eotaxin (CCL-11) are elevated in plasma of FMS patients (Zhang et al., 2008). MCP-1 is a strong mast cell chemoattractant (Conti et al., 1998) and also triggers mast cells in rodents (Conti and Theoharides, 1994). MCP-1 induced prolonged muscle hyperalgesia in rats via activation of its high-affinity receptor, CC Chemokine receptor 2 (CCR2), on the peripheral nerve terminals (Alvarez et al., 2014). Myoblasts treated with MCP-1 secreted significant amounts of the key pro-inflammatory cytokine IL-1β (Zhang et al., 2008). C-reactive protein (CRP) is now considered a marker of chronic inflammation. CRP may be useful in the diagnostic of FMS (and depression/anxiety that often accompany FMS), even though there is no direct correlation reported (De Berardis et al., 2006Orsolini et al., 2018).
Mast cells derive from the bone marrow and mature in response to SCF, which acts via the cell surface tyrosine kinase KIT receptor (Galli et al., 2011). Mast cell progenitors then migrate in all tissues. As a result, mast cell mediators can affect all organs and lead to multiple symptoms. Mast cells are found adjacent to blood vessels and nerve endings; in the brain, mast cells are located in the thalamus, hypothalamus and median eminence (Edvinsson et al., 1976;Lambracht-Hall et al., 1990;Theoharides et al., 2015d).
Finally, mast cells can release extracellular vesicles (exosomes) (Skokos et al., 2002(Skokos et al., , 2003 that could deliver regulatory molecules, including mtDNA and microRNAs (Kawikova and Askenase, 2014). Such microvesicles have been implicated in brain disorders Kawikova and Askenase, 2014) and pain disorders (Rafiee et al., 2018;Silva-Freire et al., 2019). We recently reported that extracellular vesicles are increased in the serum of children with ASD, contained mtDNA and stimulated cultured human microglia to secrete the pro-inflammatory molecules IL-1β and CXCL8 .

Mast Cell Interactions With Microglia
Mast cells communicate with microglia (Skaper et al., 2012(Skaper et al., , 2014b. Mediators secreted from mast cells, (Zhang et al., 2016) such as histamine (Dong et al., 2014) and tryptase,  can activate microglia leading to secretion of the proinflammatory cytokines IL-1β, IL-6 and TNF. Microglia can also be activated by CRH secreted from mast cells (Wang et al., 2002;Kempuraj et al., 2004). Stimulation of brain mast cells in mice led to activation of microglia, which was decreased by administration of a mast cell inhibitor (Dong et al., 2017).

FUTURE DIRECTIONS
Research should focus on identifying in serum of patients with FMS novel molecules that are involved in pain transmission such as bradykinin, CGRP and IL-31. Extracellular vesicles should also be isolated from the serum and CSF of FMS patients, their content identified, and their effect investigated on cultured human mast cells and microglia. Such possible interactions would serve as useful in vitro assays for the screening of potential novel treatment agents. Recent reports have also stressed the possible use of the cytokine IL-37, (Mastrangelo et al., 2018) which is known to have anti-inflammatory actions (Cavalli and Dinarello, 2018). It would be important to explore the possible use of IL-37 isoforms in the treatment of FMS.

AUTHOR CONTRIBUTIONS
TT, IT, and MB participated in searching the literature. TT and IT wrote or contributed to the writing of the manuscript. IT prepared the figure.
FUNDING Some aspects of our work described were supported in part by the National Institutes of Health (NIH) (Grants NS38326 and AR47652), as well as the Michael and Katherine Johnson Family Fund to TT.