3.3. Biomarkers of Inflammation
Of the three interleukins we chose to study, IL1β and IL21 were significantly elevated compared with normal values, whereas IL10 was within normal limits (Table 2
). Tumor necrosis factor alpha (TNFα), interferon gamma (INFγ), and CD30 were also elevated compared to normal values (Table 2
). P-selectin and monocyte chemoattractant protein-1 (MCP-1) were within normal limits, but soluble CD40 ligand (sCD40L/CD154) and RANTES (CCL5) were found to be at significantly lower values than normal (Table 2
). There were no correlations of cytokines/chemokines with autoantibodies (Table 3
), but many cytokine/chemokine correlations were identified using Pearson’s correlation coefficients, highlighted in red font in Table 4
In this study, we evaluated the hypothesis that a cytokine or chemokine might be identified related to platelet activation to explain the observation that POTS patients seem to have a significant association with δ-SPD. We found that the majority of our POTS patients had platelet δ-SPD, consistent with findings we have previously reported [7
]. We also identified significant elevations of IL1β, IL21, TNFα, INFγ, and CD30 in the plasma of POTS patients with elevated autoantibodies to at least the A1 adrenergic receptor. These five biomarkers of inflammation are all mediators of the innate immune system. Thus, POTS patients appear to have evidence of an ongoing inflammatory process in addition to the previously described autoimmune process.
A recent review article by Mantovani et al. (2019) describes that the Interleukin-1 family is composed of 11 soluble molecules and 10 receptors, divided into subgroups based upon molecular sequence and activity including agonist activity, receptor antagonists, and an anti-inflammatory cytokine [41
]. They conclude that IL-1 represents a paradigm for inflammation and immunity as a metanarrative of 21st century medicine; this could also apply to our understanding of POTS, with reported evidence of autoantibodies as a potential etiology of the disorder.
Leukocytes, including monocytes, macrophages, neutrophils, and dendritic and endothelial cells, are the major source of IL-1β, produced by the caspase cleavage of a pre-protein [42
]. Tunjungputri, et al. (2018) found increased plasma levels of IL-1β that were positively associated with platelet numbers, p-selectin expression, and several platelet single-nucleotide polymorphisms [30
]. IL-1β is an essential cytokine in innate immunity as one of the first signals in plasma in response to infection [29
]. IL-1β has been reported to be highly involved with autoimmune and autoinflammatory diseases; the pathogenesis of autoimmune disease involves genetic susceptibility [43
]. Classic autoimmune disorders are characterized by the presence of autoantibodies and autoantigen-specific T cells [44
]. The symptoms of autoimmune disease and autoinflammatory disorders overlap significantly. Autoinflammatory diseases are characterized by innate immunity abnormalities, usually without infections and without autoantibodies [45
]. Autoimmune disorders are propelled by type I interferon, whereas autoinflammation is distinguished by elevations of inflammasome-induced IL-1β and IL-18; IL-1β and type I IFN counter-regulate one another and interfere with adaptive immune responses [45
]. IL-1β mediates immunity for both innate and adaptive responses; it promotes innate immunity via the recruitment of inflammatory cells, whereas it enhances T cell differentiation for adaptive immunity [48
]. Since we have identified elevations of both autoantibodies against adrenergic and muscarinic receptors in our study group, the approximate 30-fold elevation of IL-1β (Table 3
) in these patients supports published data that suggest POTS is an autoimmune disorder, presumably via the persistent activation of T cells. A schematic diagram of the cytokine/chemokines described in this manuscript and interactions with immune cells is available as Supplementary Figure S3
The IL-21 levels were elevated approximately 10-fold (Table 4
) in these patients. IL-21 is produced by CD4 + T cells, natural killer T cells (NKT), and follicular helper cells and induces B cell proliferation and differentiation into plasma cells [42
]. IL-21 can also be immunosuppressive because of its ability to induce IL-10 [49
]. Elevations of IL-21 have been reported in the pathogenesis of some autoimmune diseases, including celiac disease (CD), rheumatoid arthritis (RA), and systemic lupus erythematous (SLE) [50
Tumor necrosis factor alpha (TNFα) was approximately 100-fold elevated in our patients (Table 4
); it is an extremely important signaling cytokine of the innate immune system that is involved in acute phase reactions and produced primarily by activated macrophages [42
]. It is also produced by T helper and NKT cells in response to IL-1; has antiviral properties [52
]; and is implicated in major depression, which is a common comorbidity in POTS [53
]. It has recently been reported that TNFα induces the inflammasome-independent production of IL-1β, causing autoimmunity [48
]. With elevations of IL-1β, IL-21, and TNFα, there is a strong implication that the development of autoantibodies in our patients may be T-cell-mediated.
Unfortunately, we did not measure the plasma levels of interferons alpha and beta (INFα/β, type I INF), which are produced by monocytes and fibroblasts and considered “non-immune interferons” [54
]. We did identify 40-fold elevations (Table 4
) in INF gamma (INFγ, type II INF) that is produced by cytotoxic and helper T cells and can activate macrophages and NKT cells, both known to be elevated in viral infections [55
]. We chose INFγ, a type II interferon, specifically thinking of a potential viral etiology for POTS, and this is a significant limitation of our study. Type I interferons have become increasingly recognized as important cytokines in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematous (SLE) [56
We included measurements of CD30, a transmembrane receptor expressed by activated T and B cells; it is part of the TNF receptor superfamily [58
]. CD30 expression has been reported in many autoimmune diseases, including RA [59
], and is thought to have regulatory effects to inhibit autoimmunity [60
]; it is also elevated in viral infections [58
]. The 20-fold elevation (Table 4
) in our POTS patients might be regulatory in an attempt to inhibit the autoimmunity, or it might be related to a recent or chronic viral infection. This implicates potential pathways for POTS, similar to those in the etiology of RA.
Two of the inflammation biomarkers we assayed had significantly lower plasma levels than normal. CD40 ligand (CD40L) is a transmembrane molecule anchored in activated T cell and platelet membranes [61
]. It can be cleaved into soluble CD40L (sCD40L), and both are important mediators of inflammation and immunity. Elevations of sCD40L have been associated with morbid obesity; cardiovascular problems; and autoimmune disorders, including RA and SLE [62
]. We were unable to identify a specific disorder associated with decreased plasma levels via a thorough literature review. It might be possible that platelet δ-SPD is an acquired condition related to chronic inflammation, and the platelets cannot synthesize nor store CD40L, resulting in the decrease in the plasma.
We also found that RANTES (CCL5) was significantly lower than normal values; this was unexpected. RANTES is a strong biomarker of platelet activation and a potent mediator of inflammation. It is reported to be elevated in numerous diseases. Our results of decreased RANTES in POTS patients do not support our hypothesis that platelets are activated in POTS. RANTES is also secreted by activated monocytes, but not to the extent of activated platelets. A question remains regarding the decreased RANTES in our patients’ plasma; our results suggest that neither platelets nor monocytes are activated, therefore RANTES is not secreted, which would elevate the substance. This result is unexpected and of unknown significance.
The limitations of our study include many of the deficiencies of a descriptive, proof of concept study. These include the lack of a case control study, a low number of subjects (34 in this report), and an incomplete number of cytokines/chemokines utilized. We did not include assessments of IL-6, IL-8, IL-17, IL-18, CD40, and type I IFNα/β. These will be included in a future case control study to validate our findings. This is not a review in immunology, but we have included discussions of cytokine/chemokine sources and their respective functions in an attempt to rationalize our findings, as five of the inflammatory biomarkers were elevated, three were within normal limits, and two unexpectedly reduced (Table 4
). An additional problem of data interpretation includes that there are no established standardized normal ranges and cut-off values for the cytokines/chemokines that we assayed. As above, a planned case control study will allow us to establish our own cut-off values. Our Quantibody®
multiplex ELISA assay provided a quality control standardized curve to determine concentrations with the lower limit of detection established by RayBiotech. We reviewed as many manuscripts as we could identify with control group biomarker ranges and scoured ranges from most of the large reference laboratory websites to find a reasonable normal range of expected biomarker concentrations we could use for our results. Finally, we did not record the therapeutic drug history and nor was BMI recorded for these patients. All the patients have been de-identified and we cannot review their medical records.
Our patients demonstrated elevations of five biomarkers of inflammation (IL-1β, IL-21, TNFα, INFγ, and CD30) that are known to be elevated in autoimmune disorders such as RA and SLE [64
]. The development of autoantibodies in POTS could potentially be due to a dysregulation of an interleukin, such as is seen in the pathogenesis of SLE with elevations of IL-21, TNF, and INFγ, as observed in our patients [65
Future plans include a comprehensive case control study to confirm our previously reported finding of autoantibodies against both adrenergic and muscarinic receptors in POTS patients. We also plan to confirm the results of this report but will include additional cytokines and chemokines, as mentioned above. Of particular interest is addressing the etiology of δ-SPD, which is likely to be an acquired condition rather than an inherited comorbidity.
The innate immune system, including the platelet, is important in defense against viral infections. Many autoimmune diseases have been hypothesized to evolve from an antecedent viral infection, and there are also a few reports that suggest that vaccinations may, on occasion, induce POTS [66
]. Other studies have reported that viral infections and molecular mimicry are likely associated with autoimmune diseases [68
]. A number of recent reports have identified POTS as a sequela of COVID-19 infection [70
]. One might postulate that molecular mimicry might induce antibodies that target adrenergic and cholinergic muscarinic receptors via receptor stoichiometry similar to an unknown antigenic epitope seen in infection and/or inflammation. A significant number of our patients have described Epstein Barr virus infections and gastrointestinal pain that could be related to an enteric viral infection preceding the onset of symptoms and ultimately the development of POTS. It may also be that the gastrointestinal pain is related to the hypomotility of the intestinal tract due to a deficiency of serotonin available to drive contraction of smooth muscle cells. Irritable bowel syndrome (IBS), a common co-morbidity in POTS, is possibly linked to the reduced secretion of serotonin by enterochromaffin cells in the gut. Enterochromaffin cells can be enhanced or attenuated by the secretory products of immune cells, such as CD4 + T cells, and patients with IBS have fewer serotonin-producing cells in the large intestine than normal control subjects [73
]. The platelet stores 99% of the serotonin outside of the central nervous system in dense granules, and more than 80% of our patients have a deficiency of platelet dense granules. We did not have evidence of platelet activation to explain the δ-SPD; it could be possible that the deficiency is related to low serotonin production in the gut and therefore results in fewer dense granules. Regardless, our hypothesis regarding platelet activation is incorrect.