Although there is more than a century of TAO research from which to draw, the aetiology and pathophysiology of TAO is not yet understood. It is not yet even known whether the first event in TAO is thrombosis or inflammation (angiitis) [1]. However, several studies have shown that infectious factors are a likely trigger for TAO development [2,3,4,5,6,7,8]. Notably, the clues that point to the role of infectious pathogens in TAO suggest gram-negative pathogens.
Recently, we discovered an infiltration of neutrophils and cytotoxic T lymphocytes in the SG of TAO patients [9]. In the current study, we wanted to discover if the infiltration of inflammatory cells in the SG of the patients was due to the presence of a pathogen inside the SG tissue or whether there was a reactive inflammation due to irritation of the peripheral nerves in ischaemic tissue or neural cell injuries in SG.
For this purpose, we evaluated the gene expression of HMGB1 as an inflammatory marker that can increase both due to release of DNA from injured cells (sterile inflammation) and also in response to gram-negative bacteria [10, 11]. Also, the gene expression of TLR4 was evaluated because it is a receptor of innate immunity for LPS and can increase due to infection with gram-negative bacteria [15]. Therefore, we hypothesised that, if the gene expression of both TLR4 and HMGB1 increased without change in the gene expression of TLR9 or RAGE, then an infectious pathogen, including Rickettsia, could be inside the SG tissue and be responsible for the inflammation in the sympathetic ganglia. If only the gene expression of TLR4 increased without any change in the gene expression of HMGB1, the infectious pathogen might be a chlamydia-related organism, in which there is a serological cross-reactivity between Rickettsia and chlamydia [16]. If HMGB1 increased without any increase in TLR4 gene expression but with an increase of TLR9 or RAGE expression, this would demonstrate an endogenous source of inflammation in the SG, probably following cell injuries that lead to DNA release [17]. However, if HMGB1 increased without any change in the gene expression of TLR4, TLR9, or RAGE, then it might be induced by the retrograde ischaemic injury of the peripheral nerves of the extremities [18]. Finally, if the gene expression of all studied genes—including TLR4, TLR9, RAGE, and HMGB1—increased, this could indicate the role of neutrophil extracellular traps (NETs) as a response to a gram-negative bacteria trigger, which could be the source of extracellular DNA for induction of sterile inflammation (infectious induced autoimmunity) [19].
Our results demonstrated that the gene expression of HMGB1 and TLR9 increased about 25- and 2-fold changes in the SG of TAO patients, respectively. However, there was no change in the gene expression of TLR4 or RAGE. Therefore, the most appropriate conclusion was that the inflammation inside the SG could be a sterile inflammation due to the presence of nucleic acids, such as DNA, but not due to ischaemic injuries of the nerve end terminals.
Next, we will explain how the possible injuries of the cells in the SG of TAO patients may lead to the release of DNA, increase the gene expression of HMGB1, and also activate the TLR9 pathway (Fig. 2).
One of the most important and dangerous components of cigarettes is cadmium [20]. Cadmium in the soil can be accumulated in tobacco leaves [21, 22]. Notably, cadmium can accumulate in the nervous system, including in the SG [23, 24]. Cadmium tends to influence mitochondrial DNA more than nuclear DNA by mtDNA cleavage, inhibit adenosine triphosphate (ATP) synthesis, and, consequently, induce apoptosis [25, 26]. Moreover, mtDNA damage, the reactive oxidative stress that is induced by cigarette smoking, enhances the release of mtDNA into the cytoplasm and, consequently, induces the expression of HMGB1 to make an HMGB1-mtDNA complex that activates cytosolic TLR9 [27]. Following TLR9 activation, interleukin 8 (IL-8), a neutrophil chemoattractant, is released, which may explain the neutrophil infiltration in the SG of the patients [9, 25].
However, if HMGB1 makes a complex with extracellular DNA due to cellular damage, this complex will require RAGE for internalisation and activation of TLR9 [28]. Owing to the fact that the gene expression of RAGE was almost unchanged in the current study, mtDNA may play an important role in initiating inflammation by activating TLR9, since mtDNA is present in cytosol and does not need to internalisation by RAGE.
In addition, according to animal-based studies, HMGB1 can be upregulated due to retrograde ischaemic injury of the peripheral nerves of the extremities or due to response to neuropeptide Y, which is usually overexpressed in the sympathetic system following psychological stress [18, 29]. Therefore, the 25-fold increase in the gene expression of HMGB1 may have several triggers, including release of mtDNA, mental stress, or ischaemic injury of the nerves due to vascular occlusion. The hypothesis.
However, aside from findings concerning the underlying trigger of inflammation in the SG of TAO patients, one of the precise findings of this study was the high gene expression of HMGB1 in the SG of TAO patients. HMGB1 is a pro-angiogenic factor, which can induce the production of vascular endothelial growth factor (VEGF) [16]. Since it has been found that the corkscrew collaterals in TAO arteriography are vaso-nervorum, not vaso-vasorum [30], the high expression of HMGB1 may be responsible for the formation of corkscrews in TAO.
Moreover, owing to the fact that HMGB1 induces both chemotaxis of neutrophils and inflammatory cells and platelet activation [31, 32], it is not yet known whether HMGB1 could be transported alongside the sympathetic nerves and released from their end terminals. Because a high serum level of HMGB1 in TAO patients has also been reported [33], it is possible that HMGB1 does not release from the end terminals of the sympathetic nerves. Instead, the possible release of neuropeptide Y due to psychological stress may induce expression of HMGB1 in the macrophages of the tissues receiving the sympathetic nerve supply [34]. Thus, it could be responsible for inflammation, thrombus formation at the site of vascular denervation, and corkscrew formation.
On the other hand, TAO might be a type of neurogenic inflammation. This hypothesis could explain the skip lesions and corkscrews in the arteriography of TAO patients. It could also explain the close relationship of the patients’ clinical signs and symptoms with smoking. In addition, it could explain TAO flare-up after mental stress and signs and symptoms related to vasoconstriction, including cold sensation of the toes, cold sensitivity, and Raynaud’s phenomenon, as the early signs of TAO. Moreover, it seems there is a close interaction between nervous stress and inflammation with consequent vascular damages [35]. However, more investigation is needed to prove or disprove this hypothesis.