Mast cell activation: beyond histamine and tryptase.

INTRODUCTION: Mast cells are found in all tissues and express numerous surface receptors allowing them to sense and respond to allergic, autoimmune, environmental, neurohormonal, pathogenic and stress triggers. Stimulated mast cells are typically called 'activated' but the mechanisms involved and the mediators released can vary considerably. Mast cell activation diseases (MCADs) include primary, secondary and idiopathic conditions, especially mast cell activation syndrome (MCAS), but mast cells are activated in many other disorders making the diagnosis and treatment challenging. AREAS COVERED: Mast cells can release numerous biologically active mediators, some of which are prestored in secretory granules while others are newly synthesized and released without degranulation. Most of the emphasis has so far been on secretion of histamine and tryptase, which do not explain all the multisystemic symptoms experienced by patients with MCADs. As a result, drug development has focused on antiproliferative therapy or blocking the action of individual mediators and not on inhibitors of mast cell activation. EXPERT OPINION: Activated mast cells are involved in the pathogenesis of MCADs, but also in other disorders making appropriate diagnosis and treatment challenging. The definition of mast cell activation should be expanded beyond histamine and tryptase, with an emphasis on better detection and treatments.

Genetics of COVID-19 and myalgic encephalomyelitis/chronic fatigue syndrome: a systematic review.

COVID-19 and ME/CFS present with some similar symptoms, especially physical and mental fatigue. In order to understand the basis of these similarities and the possibility of underlying common genetic components, we performed a systematic review of all published genetic association and cohort studies regarding COVID-19 and ME/CFS and extracted the genes along with the genetic variants investigated. We then performed gene ontology and pathway analysis of those genes that gave significant results in the individual studies to yield functional annotations of the studied genes using protein analysis through evolutionary relationships (PANTHER) VERSION 17.0 software. Finally, we identified the common genetic components of these two conditions. Seventy-one studies for COVID-19 and 26 studies for ME/CFS were included in the systematic review in which the expression of 97 genes for COVID-19 and 429 genes for ME/CFS were significantly affected. We found that ACE, HLA-A, HLA-C, HLA-DQA1, HLA-DRB1, and TYK2 are the common genes that gave significant results. The findings of the pathway analysis highlight the contribution of inflammation mediated by chemokine and cytokine signaling pathways, and the T cell activation and Toll receptor signaling pathways. Protein class analysis revealed the contribution of defense/immunity proteins, as well as protein-modifying enzymes. Our results suggest that the pathogenesis of both syndromes could involve some immune dysfunction.

Exosome-associated mitochondrial DNA from patients with myalgic encephalomyelitis/chronic fatigue syndrome stimulates human microglia to release IL-1ß.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease that presents with fatigue, sleep disturbances, malaise, and cognitive problems. The pathogenesis of ME/CFS is presently unknown, and serum levels of potential biomarkers have been inconsistent. Here, we show that mitochondrial DNA (mtDNA) associated with serum exosomes, is increased in ME/CFS patients only after exercise. Moreover, exosomes isolated from patients with ME/CFS stimulate significant release of IL-1ß from cultured human microglia. These results provide evidence that activation of microglia by serum-derived exosomes may serve as a potential novel pathogenetic factor and target for treatment of ME/CFS.

Calprotectin and Imbalances between Acute-Phase Mediators Are Associated with Critical Illness in COVID-19.

The trajectory from moderate and severe COVID-19 into acute respiratory distress syndrome (ARDS) necessitating mechanical ventilation (MV) is a field of active research. We determined serum levels within 24 h of presentation of 20 different sets of mediators (calprotectin, pro- and anti-inflammatory cytokines, interferons) of patients with COVID-19 at different stages of severity (asymptomatic, moderate, severe and ARDS/MV). The primary endpoint was to define associations with critical illness, and the secondary endpoint was to identify the pathways associated with mortality. Results were validated in serial measurements of mediators among participants of the SAVE-MORE trial. Levels of the proinflammatory interleukin (IL)-8, IL-18, matrix metalloproteinase-9, platelet-derived growth factor (PDGF)-B and calprotectin (S100A8/A9) were significantly higher in patients with ARDS and MV. Levels of the anti-inflammatory IL-1ra and IL-33r were also increased; IL-38 was increased only in asymptomatic patients but significantly decreased in the more severe cases. Multivariate ordinal regression showed that pathways of IL-6, IL-33 and calprotectin were associated with significant probability for worse outcome. Calprotectin was serially increased from baseline among patients who progressed to ARDS and MV. Further research is needed to decipher the significance of these findings compared to other acute-phase reactants, such as C-reactive protein (CRP) or ferritin, for the prognosis and development of effective treatments.

Could SARS-CoV-2 Spike Protein Be Responsible for Long-COVID Syndrome?

SARS-CoV-2 infects cells via its spike protein binding to its surface receptor on target cells and results in acute symptoms involving especially the lungs known as COVID-19. However, increasing evidence indicates that many patients develop a chronic condition characterized by fatigue and neuropsychiatric symptoms, termed long-COVID. Most of the vaccines produced so far for COVID-19 direct mammalian cells via either mRNA or an adenovirus vector to express the spike protein, or administer recombinant spike protein, which is recognized by the immune system leading to the production of neutralizing antibodies. Recent publications provide new findings that may help decipher the pathogenesis of long-COVID. One paper reported perivascular inflammation in brains of deceased patients with COVID-19, while others showed that the spike protein could damage the endothelium in an animal model, that it could disrupt an in vitro model of the blood-brain barrier (BBB), and that it can cross the BBB resulting in perivascular inflammation. Moreover, the spike protein appears to share antigenic epitopes with human molecular chaperons resulting in autoimmunity and can activate toll-like receptors (TLRs), leading to release of inflammatory cytokines. Moreover, some antibodies produced against the spike protein may not be neutralizing, but may change its conformation rendering it more likely to bind to its receptor. As a result, one wonders whether the spike protein entering the brain or being expressed by brain cells could activate microglia, alone or together with inflammatory cytokines, since protective antibodies could not cross the BBB, leading to neuro-inflammation and contributing to long-COVID. Hence, there is urgent need to better understand the neurotoxic effects of the spike protein and to consider possible interventions to mitigate spike protein-related detrimental effects to the brain, possibly via use of small natural molecules, especially the flavonoids luteolin and quercetin.

Immune-Related Gene Polymorphisms and Pharmacogenetic Studies in Nephrology.

A large subgroup of patients with chronic kidney disease still encounter serious adverse effects and lack of responsiveness to medications, possibly because of the interindividual genetic variability in genes involved in the metabolism and transport of the treatments used. As a consequence, several pharmacogenetic studies have been conducted in nephrology patients that examine the effect of genetic variants in response to treatment in kidney diseases. The present commentary focuses on immune-related genes (TNF [tumor necrosis factor], MIF [macrophage migration inhibitory factor], and IL-10 [interleukin 10]) or those genes that may regulate the response to immunosuppressive medications (ABCB1 [ATP binding cassette subfamily B member 1] and ITPA [inosine triphosphatase]) used in kidney diseases. These genes were selected from those showing significant results in a recent meta-analysis of pharmacogenetic studies of patients with chronic kidney disease. This commentary highlights that certain polymorphisms should be investigated in patients with kidney diseases, especially if they are to be administered immunosuppressive agents. In certain cases, flavonoids such as quercetin may be beneficial.

Long-COVID syndrome-associated brain fog and chemofog: Luteolin to the rescue.

COVID-19 leads to severe respiratory problems, but also to long-COVID syndrome associated primarily with cognitive dysfunction and fatigue. Long-COVID syndrome symptoms, especially brain fog, are similar to those experienced by patients undertaking or following chemotherapy for cancer (chemofog or chemobrain), as well in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) or mast cell activation syndrome (MCAS). The pathogenesis of brain fog in these illnesses is presently unknown but may involve neuroinflammation via mast cells stimulated by pathogenic and stress stimuli to release mediators that activate microglia and lead to inflammation in the hypothalamus. These processes could be mitigated by phytosomal formulation (in olive pomace oil) of the natural flavonoid luteolin.

COVID-19, microthromboses, inflammation, and platelet activating factor.

Recent articles report elevated markers of coagulation, endothelial injury, and microthromboses in lungs from deceased COVID-19 patients. However, there has been no discussion of what may induce intravascular coagulation. Platelets are critical in the formation of thrombi and their most potent trigger is platelet activating factor (PAF), first characterized by Demopoulos and colleagues in 1979. PAF is produced by cells involved in host defense and its biological actions bear similarities with COVID-19 disease manifestations. PAF can also stimulate perivascular mast cell activation, leading to inflammation implicated in severe acute respiratory syndrome (SARS). Mast cells are plentiful in the lungs and are a rich source of PAF and of inflammatory cytokines, such as IL-1ß and IL-6, which may contribute to COVID-19 and especially SARS. The histamine-1 receptor antagonist rupatadine was developed to have anti-PAF activity, and also inhibits activation of human mast cells in response to PAF. Rupatadine could be repurposed for COVID-19 prophylaxis alone or together with other PAF-inhibitors of natural origin such as the flavonoids quercetin and luteolin, which have antiviral, anti-inflammatory, and anti-PAF actions.

The impact of psychological stress on mast cells.

OBJECTIVE: Atopic diseases worsen with psychological stress, but how stress contributes to their pathogenesis is still not clear. We review the evidence supporting the premise that stress contributes to allergic and inflammatory processes through stimulation of mast cells (MCs) by neuroimmune stimuli. DATA SOURCES: PubMed was searched between 1950 and 2019 using the following terms: allergies, atopic diseases, corticotropin-releasing hormone, inflammation, hypothalamic-pituitary-adrenal axis, mast cells, mastocytosis, neuropeptides, psychological stress, neurotensin, and substance P. STUDY SELECTIONS: Only articles published in English were selected based on their relevance to stress and MCs, especially those that discussed potential mechanisms of action. RESULTS: Psychological stress worsens many diseases, especially asthma, atopic dermatitis, and mastocytosis. This effect is mediated through MCs stimulated by neuropeptides, especially corticotropin-releasing hormone, neurotensin, and substance P, a process augmented by interleukin-33. CONCLUSION: Understanding how stress stimulates MCs to release proinflammatory mediators is important in advancing treatments for diseases that worsen with stress.

IL-37 is increased in brains of children with autism spectrum disorder and inhibits human microglia stimulated by neurotensin.

Autism spectrum disorder (ASD) does not have a distinct pathogenesis or effective treatment. Increasing evidence supports the presence of immune dysfunction and inflammation in the brains of children with ASD. In this report, we present data that gene expression of the antiinflammatory cytokine IL-37, as well as of the proinflammatory cytokines IL-18 and TNF, is increased in the amygdala and dorsolateral prefrontal cortex of children with ASD as compared to non-ASD controls. Gene expression of IL-18R, which is a receptor for both IL-18 and IL-37, is also increased in the same brain areas of children with ASD. Interestingly, gene expression of the NTR3/sortilin receptor is reduced in the amygdala and dorsolateral prefrontal cortex. Pretreatment of cultured human microglia from normal adult brains with human recombinant IL-37 (1 to 100 ng/mL) inhibits neurotensin (NT)-stimulated secretion and gene expression of IL-1ß and CXCL8. Another key finding is that NT, as well as the proinflammatory cytokines IL-1ß and TNF increase IL-37 gene expression in cultured human microglia. The data presented here highlight the connection between inflammation and ASD, supporting the development of IL-37 as a potential therapeutic agent of ASD.

Nasal cytology with emphasis on mast cells can improve the diagnosis and treatment of chronic rhinosinusitis.

OBJECTIVE: Chronic rhinosinusitis (CRS) involves inflammation of the nasal and para-nasal mucosa. Due to its heterogeneous nature, unknown pathogenesis, and high recurrence rate, effective treatment is difficult. Nasal cytology is presently not a part of the routine diagnosis or treatment decision for CRS. DATA SOURCES: A literature search was performed for published papers in English between January 1990 and June 2019 using MEDLINE. STUDY SELECTION: Terms used were chronic rhinosinusitis, eosinophils, etiology, immunopathology, inflammation, mast cells, nasal cytology, polyps, and treatment. Both reviews and original articles were collected and studied. RESULTS: There is no standard nasal fluid, mucus sampling, or staining techniques for identifying inflammatory cell types. Results were divergent from different countries. Moreover, the main focus of these papers on the cells in nasal washings was eosinophils, with infrequent mentioning of other cell types that may imply different etiology and pathology. The heterogeneous cell profile of CRS and the role of mast cells have been unappreciated due to the lack of specific immunohistochemical technique or study of its unique mediators. CONCLUSIONS: Nasal cytology could help distinguish the type and the activation state of inflammatory cells. Thus it can help in providing a clearer picture of CRS pathogenesis, identifying different patient groups, and developing effective treatments.

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-1ß, 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.

Effects of an Extract of Salmon Milt on Symptoms and Serum TNF and Substance P in Patients With Fibromyalgia Syndrome.

PURPOSE: The aim of this study was to evaluate the effects of a dietary supplement containing primarily an extract of salmon's milt (semen) on symptoms and blood levels of proinflammatory molecules in patients with fibromyalgia syndrome (FMS), a chronic, painful musculoskeletal disease without a distinct pathogenesis or treatment. We recently reported increased serum levels of the proinflammatory molecules substance P (SP) and tumor necrosis factor (TNF) in patients with FMS as compared to those in normal controls. METHODS: This prospective, open-label study was conducted in patients with FMS (n = 87; 80 women, 7 men; age range, 18-80 years) selected from 2 clinical centers in Spain. Patients were administered the supplement and were evaluated at weeks 1 (before treatment), 4, 8, and 12 (end of treatment) for clinical parameters of functioning, fatigue, and pain, as well as overall impression. Patients were directed to take 1 capsule per day in the morning for the first 4 weeks, followed by 1 capsule in the morning and 1 capsule in the evening for the remaining 8 weeks. Differences in symptom scores in patients with FMS between weeks 1 and weeks 4, 8, and 12 were evaluated using ANOVA. Blood was obtained and serum separated in patients with FMS at 1 and 12 weeks and in a separate population of healthy controls (n = 20; 15 women, 5 men; age range, 25-65 years). Serum levels of SP and TNF were measured in patients with FMS at 1 and 12 weeks and in healthy controls by ELISA. TNF and SP levels in patients with FMS were compared between weeks 1 and 12, as well as between patients with FMS and untreated controls, using the Mann-Whitney U test. FINDINGS: Clinical parameters of functioning, fatigue, and pain, as well as overall impression, were improved significantly at 4 weeks as compared to 1 week and remained unchanged for the duration of the study (all, P < 0.0001). Serum TNF and SP levels were significantly elevated at 1 week in patients with FMS compared to controls and were decreased significantly at 12 weeks as compared to 1 week (all, P < 0.0001). IMPLICATIONS: Our findings indicate that this dietary supplement may significantly improve symptoms in patients with FMS. This is the first time to our knowledge that any molecule has been reported to be associated with a reduction in serum SP level. Consequently, the supplement or its hypothesized main active ingredient, spermine, may be developed as a novel treatment approach to FMS or other neuroinflammatory conditions. ClinicalTrials.gov identifier: NCT03911882.

Recent advances in our understanding of mast cell activation - or should it be mast cell mediator disorders?

INTRODUCTION: An increasing number of patients present with multiple symptoms affecting many organs including the brain due to multiple mediators released by mast cells. These unique tissue immune cells are critical for allergic reactions triggered by immunoglobulin E (IgE), but are also stimulated (not activated) by immune, drug, environmental, food, infectious, and stress triggers, leading to secretion of multiple mediators often without histamine and tryptase. The presentation, diagnosis, and management of the spectrum of mast cell disorders are very confusing. As a result, neuropsychiatric symptoms have been left out, and diagnostic criteria made stricter excluding most patients. Areas covered: A literature search was performed on papers published between January 1990 and November 2018 using MEDLINE. Terms used were activation, antihistamines, atopy, autism, brain fog, heparin, KIT mutation, IgE, inflammation, IL-6, IL-31, IL-37, luteolin, mast cells, mastocytosis, mediators, mycotoxins, release, secretion, tetramethoxyluteolin, and tryptase. Expert opinion: Conditions associated with elevated serum or urine levels of any mast cell mediator, in the absence of comorbidities that could explain elevated levels, should be considered 'Mast Cell Mediator Disorders (MCMD).' Emphasis should be placed on the identification of unique mast cell mediators, and development of drugs or supplements that inhibit their release.

Mitochondrial dysfunction in affected skin and increased mitochondrial DNA in serum from patients with psoriasis.

Psoriasis is characterized by keratinocyte proliferation and chronic inflammation, but the pathogenesis is still unclear. Dysregulated mitochondria (mt) could lead to reduced apoptosis and extracellular secretion of mtDNA, acting as "innate pathogen" triggering inflammation. Serum was obtained from healthy volunteers and psoriatic patients. Mitochondrial DNA was extracted from the serum and amplified with quantitative PCR (qPCR). Punch biopsies were obtained from lesional and non-lesional psoriatic skin (10 cm apart) and from healthy volunteers, were placed in RNA later and were stored at -80°C until RNA was extracted and cDNA was synthesized; gene expression of uncoupling protein 2 (UCP2), Dynamin-related protein 1 (Drp1) and calcineurin, involved in the regulation of mitochondria function, was detected with qPCR. Mitochondrial DNA was significantly increased (7s, P = 0.0496 and Cytochrome B, CytB, P = 0.0403) in the serum of psoriatic patients (n = 63) as compared to controls (n = 27). Gene expression was significantly reduced for UCP2 (P = 0.0218), Drp1 (P = 0.0001) and calcineurin (P = 0.0001) in lesional psoriatic skin, as compared to non-lesional or control skin. Increased serum extracellular mtDNA in psoriatic patients and decreased expression of mitochondrial regulatory proteins in psoriatic skin suggest increased inflammation and reduced keratinocyte apoptosis, respectively. Inhibitors of mtDNA secretion and/or UCP2 stimulants may be potential treatment options.

Chondroitin sulfate inhibits secretion of TNF and CXCL8 from human mast cells stimulated by IL-33.

Glycosaminoglycans (GAGs) are linear, highly negatively charged carbohydrate chains present in connective tissues. Chondroitin sulfate (CS) and heparin (Hep) are also found in the numerous secretory granules of mast cells (MC), tissue immune cells involved in allergic and inflammatory reactions. CS and Hep may inhibit secretion of histamine from rat connective tissue MC, but their effect on human MC remains unknown. Human LAD2 MC were pre-incubated with CS, Hep, or dermatan sulfate (DS) before being stimulated by either the peptide substance P (SP, 2 µM) or the cytokine IL-33 (10 ng/mL). Preincubation with CS had no effect on MC degranulation stimulated by SP, but inhibited TNF (60%) and CXCL8 (45%) secretion from LAD2 cells stimulated by IL-33. Fluorescein-conjugated CS (CS-F) was internalized by LAD2 cells only at 37 °C, but not 4 °C, indicating it occurred by endocytosis. DS and Hep inhibited IL-33-stimulated secretion of TNF and CXCL8 to a similar extent as CS. None of the GAGs tested inhibited IL-33-stimulated gene expression of either TNF or CXCL8. There was no effect of CS on ionomycin-stimulated calcium influx. There was also no effect of CS on surface expression of the IL-33 receptor, ST2. Neutralization of the hyaluronan receptor CD44 did not affect the internalization of CS-F. The findings in this article show that CS inhibits secretion of TNF and CXCL8 from human cultured MC stimulated by IL-33. CS could be formulated for systemic or topical treatment of allergic or inflammatory diseases, such as atopic dermatitis, cutaneous mastocytosis, and psoriasis. © 2018 BioFactors, 45(1):49-61, 2019.

Substance P and IL-33 administered together stimulate a marked secretion of IL-1ß from human mast cells, inhibited by methoxyluteolin.

Mast cells are critical for allergic and inflammatory responses in which the peptide substance P (SP) and the cytokine IL-33 are involved. SP (0.01-1 µM) administered together with IL-33 (30 ng/mL) to human cultured LAD2 mast cells stimulates a marked increase (P < 0.0001) in secretion of the proinflammatory cytokine IL-1ß. Preincubation of LAD2 (30 min) with the SP receptor (NK-1) antagonists L-733,060 (10 µM) or CP-96345 (10 µM) inhibits (P < 0.001) secretion of IL-1ß stimulated by either SP (1 µM) or SP together with IL-33 (30 ng/mL). Surprisingly, secretion of IL-1ß stimulated by IL-33 is inhibited (P < 0.001) by each NK-1 antagonist. Preincubation with an antibody against the IL-33 receptor ST2 inhibits (P < 0.0001) secretion of IL-1ß stimulated either by IL-33 or together with SP. The combination of SP (1 µM) with IL-33 (30 ng/mL) increases IL-1ß gene expression by 90-fold in LAD2 cells and by 200-fold in primary cultured mast cells from human umbilical cord blood. The combination of SP and IL-33 increases intracellular levels of IL-1ß in LAD2 by 100-fold and gene expression of IL-1ß and procaspase-1 by fivefold and pro-IL-1ß by twofold. Active caspase-1 is present even in unstimulated cells and is detected extracellularly. Preincubation of LAD2 cells with the natural flavonoid methoxyluteolin (1-100 mM) inhibits (P < 0.0001) secretion and gene expression of IL-1ß, procaspase-1, and pro-IL-1ß. Mast cell secretion of IL-1ß in response to SP and IL-33 reveals targets for the development of antiinflammatory therapies.