Although many important genes may remain undetected or overlooked, we have endeavored to highlight several pathways, as well as individual genes, that likely contribute to CIRS-ciguatera using data predominantly from our HS results of 55 probes.
Coagulopathies and hemostasis
Previous functional genomics studies in mice have shown disruption in the complement and coagulation pathways after an acute exposure to ciguatoxin . Additionally, human cases of CIRS-ciguatera have been identified with similar abnormalities, such as elevated complement C4a, irregularities in VWF and Factor VIII, and elevated levels of plasminogen activator inhibitor-1 . The HS gene set identified in the current study contains VWF, coagulation factor XIII, thrombospondin (CD36) and CD9. These genes function in platelet activation, which was one of the most enriched pathways revealed by DAVID GO analysis. CD9 is a member of the tetraspanin family and one of the most abundant proteins on the platelet cell membrane . It was determined to be down-regulated here by microarray (1.9 fold) and validated by qPCR (1.8 fold), and it has long been known to play a role in cell motility and adhesion . An interesting aspect of CD9 in the case of CIRS-ciguatera is that it influences the expression of MMP9, which is found up-regulated at the protein level in the plasma of over 60% of CIRS-ciguatera cases. In keratinocytes, down-regulation of CD9 was found to increase activity and expression of MMP9 through JNK signaling , while in a fibrosarcoma cell line, epithelial growth factor receptor, EGFR, was an important intermediary for increased release of pro-MMP9 by CD9 . CD9 is critical for platelet microparticle release, which are formed from budding of the cytoplasmic membrane and are an order of magnitude larger than exosomes . Roughly 70–90% of all microparticles in the bloodstream originate from platelets . They are thought to be part of a wide-ranging form of inter-cellular communication that function by carrying bioactive components and signaling molecules, which can be released into target cells, in many instances, altering the function of those cells . In addition to their function in coagulation, the microparticles are also implicated in a range of inflammatory and autoimmune diseases [22,23]. A recent study identified another high density platelet receptor, CD36 (aka, thrombospondin receptor), which was similarly down-regulated in these CIRS-ciguatera patients (1.56 fold), that strongly co-localized with CD9, not only on the surface of platelets, but also on dermal microvessel endothelial cells, possibly forming sites for platelet adhesion and aggregation . These receptors, together with the genes for VWF and Factor XIII, which were also down-regulated in this study, most likely play a role in the coagulopathies and hemodynamics associated with CIRS-ciguatera. What is also interesting is that one of the most common Folk tests for ciguatoxic fish used in the South Pacific is called the bleeder test. This test relies on hemorrhaging of an incision in the tail of the fish to indicate toxicity .
Long non-coding RNA (lnc-RNA)
Several lncRNAs were found to be differentially expressed in this study, leading us to believe that these regulators of transcription and translation play an important role in CIRS-ciguatera. Non-coding RNA (ncRNA) research is a relatively new field and, although microRNA research far outpaces lncRNA, its importance is becoming noteworthy as some single lncRNA knockouts have proven lethal in mice . Relatively little is known about the functions of most lncRNAs; however, a few species have been characterized because of the significance of their functions. One such species was found to be up-regulated 1.66 fold in CIRS-ciguatera patients, Nuclear Enriched Abundant Transcript 1, NEAT1. NEAT1 is a critical component of nuclear paraspeckles, a nuclear body found to have a role in sequestering certain RNA transcripts in the nucleoplasm, thus keeping them from being translated into proteins in the cytoplasm. It is thought that this complex binds transcripts through their edited 3′ UTR that is then cleaved under a stress response, which allows the transcripts to be sent immediately to the cytoplasm for translation . In a different paradigm of transcriptional control, up-regulation of NEAT1 was described in response to viral infection and Toll receptor pathways, which in turn led to increased binding and sequestration of IL-8 transcriptional repressors by the paraspeckle, thus allowing increased expression of the cytokine . Early work identified a smaller form of NEAT1 in trophoblasts that could suppress gene expression of the major histocompatability complex (MHC) through sequestration of a critical transcription factor, signal transducer and activator of transcription 1 (STAT1), although in this instance, STAT1 was sequestered in the cytoplasm instead of the nucleus . This fetal isoform of NEAT1 was not found expressed in adult tissues, which is interesting because several MHC genes were significantly down-regulated in our patient samples while STAT1 was significantly up-regulated 1.42 fold.
MHC and adaptive immunity
Adaptive immunity involves the destruction of foreign pathogens and presentation of their peptide remains to T cells to begin the processes of antibody production by B cells, and “attack on site” natural killer (NK) cells and cytotoxic T cells. The direct killing of cells usually involves the emptying of lytic enzymes, granzymes (GZM), onto targeted cells. Until recently, it was thought that these cells acted exclusively as part of the innate immune response. However, new evidence has shown that these cells also shape adaptive responses and can play a role in autoimmune disorders, such as Multiple Sclerosis, where CD56bright NK cells were shown to kill activated autologous T cells through GZM-K (up-regulated 1.54 fold here) dependent mechanisms. GZMK has also been suggested as a marker for different stages of sepsis . The heterodimeric HLA-DQ protein, composed of an alpha and beta subunit, acts as an antigen-presenting molecule of foreign peptide fragments, as opposed to MHC class I proteins that present self molecules. Several probes for the MHC class II gene HLA-DQB were found down-regulated in CIRS-ciguatera patients. In earlier work, we discovered that certain DRB and DQB class II HLA haplotypes were more susceptible to chronic illness caused by acute exposure to ciguatoxins . This is not unusual in chronic inflammatory/autoimmune illnesses. In fact, the greatest risk factor for Type I diabetes in Caucasians and African Americans was found to be certain HLA-DQ genotypes , as was DRB-DQ genotypes in late autoimmune diabetes in adults (LADA) . Additionally, two DQ haplotypes are linked with nearly all celiac disease (CD) , the DQ2 haplotype, which is present in more than 95% of CD, and DQ8, which is present in the remainder. These two haplotypes are also over-represented in many other inflammatory disorders, including Addison’s disease, Hashimotos thyroiditis, Graves’ disease, Sjogren’s syndrome and others (for review see ).
Antigen presenting cells (APC) display foreign peptide fragments through MHC receptors, these fragments are then probed by T cells through the T cell receptor (TCR). The TCR is a heterodimer, typically composed of alpha and beta subunits, with each subunit containing a variable region that interacts with the APC presented peptide. Out of a possible 46 TCR beta variable (TCRBV) probes (coding for 45 genes), our LS set of 193 significant probes contained six, all up-regulated from 1.51 to 2.33 FC. T cells not only combat infectious pathogens, they are also important in the surveillance of autoreactive self antigens that can result in autoimmune syndromes or cancer. The gene CD274, aka programmed cell death ligand 1 (PDL1, or B7-H1), up-regulated 1.7 fold here, is important for maintaining peripheral tolerance and is a powerful suppressor of T cell-mediated immunity. Over expression of CD274 by tumors, or due to chronic infection allows immune avoidance through T cell anergy, exhaustion, apoptosis and other mechanisms, and high levels of the receptor have been correlated with poor prognoses for certain cancers . The up-regulation of TCRs and PDL1 seem to indicate an attempt to generate T cells clones for a specific antigen.
Our findings are replete with genes involved in inflammation, and although a simple explanation of how this differential expression accounts for CIRS is impossible, there are too many important inflammatory threads to ignore. Activation of inflammation is well documented through Toll-like receptors (TLR) and Interleukin-1 receptor like receptor (ILR) signaling. The single immunoglobulin and IL-1 receptor (SIGIRR), found up-regulated in our patients 1.55 fold, is implicated in infectious and sterile inflammation, as well as autoimmune disease. It is also known to blunt the signaling of both TLR and IL-1, in addition to other interleukins such as IL-18 (for review see ). The IL-18 receptor is an alpha-beta heterodimer, and the beta subunit, IL-18RAP, was up-regulated in patients 1.4 fold. This subunit is also part of a linkage disequilibrium block found in CD, and its mRNA expression in blood is an important disease correlate . VIP was shown to down-regulate TLRs while at the same time up-regulating SIGIRR expression . Since over 90% of CIRS-ciguatera patients have findings of abnormally low VIP in plasma , the down-regulation of the VIP receptor found here, VIPR2 (1.7 fold), would seemingly further diminish the anti-inflammatory effects of what little neuropeptide is circulating in the blood. Toll interacting protein (TOLLIP), down-regulated in patients 1.77 fold, is an inhibitor of TLR signaling and has also been shown to modulate both IL-1 and LPS inflammatory pathways, as well as TNFα and IL-6 gene expression . Its role as an immune regulator was further expanded when it was found to inhibit TGFβ signaling , which could prove important in CIRS-ciguatera because TGFβ-1 is abnormally high in over 90% of cases.
Because ciguatoxins are toxic in minute amounts, their detection in tainted fish or exposed patients is extremely difficult, and the diagnosis of CFP relies predominantly on symptoms following a fish meal. Abnormalities in blood proteomic measurements of MSH, VIP, C4a, TGFβ-1, MMP9, ADH/osmolality, ACTH/cortisol and/or VEGF are routinely found in the chronic syndrome, but we do not know if these anomalies are detectable at the acute stage. Additionally, these proteomic abnormalities are not unique to CFP, but are also found in patients with other biotoxin-induced CIRS such as toxic mold exposure  and acute Lyme disease . The concept of a “final common pathway” of chronic immune activation seen in these diverse syndromes is consistent with the presentation of a multisystem illness that is not defined by any single symptom or group of symptoms, or unique proteome with our current testing. This increases the importance of identifying a genomic fingerprint for these different illnesses to provide objective support for use in differential diagnosis. Additionally, since the proteome of whole blood is not simply the product of white blood cells, but is more so the product of all the organs being supplied, there is no tidy explanation to correlate gene expression with the proteomic abnormalities we have recorded. However, the benefit of this paradigm in whole blood is that while the genomic research interrogates the leukocytes, the proteomics can interrogate the entire body. Further research is needed to clarify this dichotomy of genomic responses in white cells separately from the proteome of blood in inflammatory illnesses.