Category: Immune System

The effect of IL-2 stimulation and treatment of TRPM3 on channel co-localisation with PIP 2 and NK cell function in myalgic encephalomyelitis/chronic fatigue syndrome patients

Abstract:

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious multifactorial disorder. The origin remains ambiguous, however reduced natural killer (NK) cell cytotoxicity is a consistent immunological feature of ME/CFS. Impaired transient receptor potential melastatin 3 (TRPM3), a phosphatidylinositol dependent channel, and impaired calcium mobilisation have been implicated in ME/CFS pathology. This investigation aimed to examine the localisation of TRPM3 at the NK cell plasma membrane and co-localisation with phosphatidylinositol 4,5-bisphosphate (PIP2). The effect of IL-2 priming and treatment using pregnenolone sulfate (PregS) and ononetin on TRPM3 co-localisation and NK cell cytotoxicity in ME/CFS patients and healthy controls (HC) was also investigated.

Methods: NK cells were isolated from 15 ME/CFS patients and 15 age- and sex-matched HC. Immunofluorescent technique was used to determine co-localisation of TRPM3 with the NK cell membrane and with PIP2 of ME/CFS patients and HC. Flow cytometry was used to determine NK cell cytotoxicity. Following IL-2 stimulation and treatment with PregS and ononetin changes in co-localisation and NK cell cytotoxicity were measured.

Results: Overnight treatment of NK cells with PregS and ononetin resulted in reduced co-localisation of TRPM3 with PIP2 and actin in HC. Co-localisation of TRPM3 with PIP2 in NK cells was significantly reduced in ME/CFS patients compared with HC following priming with IL-2. A significant increase in co-localisation of TRPM3 with PIP2 was reported following overnight treatment with ononetin within ME/CFS patients and between groups. Baseline NK cell cytotoxicity was significantly reduced in ME/CFS patients; however, no changes were observed following overnight incubation with IL-2, PregS and ononetin between HC and ME/CFS patients. IL-2 stimulation significantly enhanced NK cell cytotoxicity in HC and ME/CFS patients.

Conclusion: Significant changes in co-localisation suggest PIP2-dependent TRPM3 function may be impaired in ME/CFS patients. Stimulation of NK cells with IL-2 significantly enhanced cytotoxic function in ME/CFS patients demonstrating normal function compared with HC. A crosstalk exists between IL-2 and TRPM3 intracellular signalling pathways which are dependent on Ca2+ influx and PIP2. While IL-2R responds to IL-2 binding in vitro, Ca2+ dysregulation and impaired intracellular signalling pathways impede NK cell function in ME/CFS patients.

Source: Eaton-Fitch N, Cabanas H, du Preez S, Staines D, Marshall-Gradisnik S. The effect of IL-2 stimulation and treatment of TRPM3 on channel co-localisation with PIP2 and NK cell function in myalgic encephalomyelitis/chronic fatigue syndrome patients. J Transl Med. 2021 Jul 15;19(1):306. doi: 10.1186/s12967-021-02974-4. PMID: 34266470.  https://pubmed.ncbi.nlm.nih.gov/34266470/

Hypothalamic-Pituitary autoimmunity and related impairment of hormone secretions in chronic fatigue syndrome

Abstract:

Context: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a severe chronic illness which reduces the quality of life. A potential role of neuroendocrine autoimmune dysfunction has been hypothesized.

Objective: To investigate the occurrence of anti-pituitary (APA) and anti-hypothalamic (AHA) antibodies and possible related hypothalamic/pituitary dysfunctions in ME/CSF patients.

Design, setting, patients and other participants: This is a case-control study conducted in University Hospital setting (Stanford, Naples). Thirty women with ME/CSF (Group 1) diagnosed according to Fukuda, Canadian, and IOM criteria, at Stanford University, were enrolled and compared with 25 age-matched healthy controls.

Main outcome measures: APA and AHA were detected by immunofluorescence; moreover, we investigated hormonal secretions of anterior pituitary and respective target glands and plasma and urinary osmolality. Both APA and AHA titers were assessed and the prevalence of pituitary hormone deficiencies was also investigated.

Results: Patients in Group 1 showed a high prevalence of AHA (33%) and APA (56%) and a significant lower levels of ACTH/cortisol, and GH peak/IGF1 vs controls (all AHA/APA negative). Patients in Group 1A (13 patients positive at high titers, ≥1:32) showed ACTH/cortisol and GH peak/ IGF1 levels significantly lower and more severe forms of ME/CFS with respect to patients in Group 1B (7 positive at middle/low titers,1:16-1:8) and 1C (10 Ab negative patients).

Conclusions: Both AHA and/or APA at high titers associated with hypothalamic/pituitary dysfunction suggest that hypothalamic/pituitary autoimmunity may play an important role in the manifestations of ME/CFS, especially in its more severe forms.

Source: De Bellis A, Bellastella G, Pernice V, Cirillo P, Longo M, Maio A, Scappaticcio L, Maiorino MI, Bellastella A, Esposito K, Montoya JG. Hypothalamic-Pituitary autoimmunity and related impairment of hormone secretions in chronic fatigue syndrome. J Clin Endocrinol Metab. 2021 Jul 13:dgab429. doi: 10.1210/clinem/dgab429. Epub ahead of print. PMID: 34254637. https://pubmed.ncbi.nlm.nih.gov/34254637/

Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterized by fatigue and post-exertional malaise, accompanied by various signs of neurological and autonomic dysfunction. ME/CFS is often triggered by an infectious episode and associated with an aberrant immune system. Here we report that ME/CFS is a disorder characterized by skewed B cell receptor gene usage. By applying a next-generation sequencing to determine the clone-based IGHV/IGHD/IGHJ repertoires, we revealed a biased usage of several IGHV genes in peripheral blood B cells from ME/CFS patients. Results of receiver operating characteristic (ROC) analysis further indicated a possibility of distinguishing patients from healthy controls, based on the skewed B cell repertoire. Meanwhile, B cell clones using IGHV3-30 and IGHV3-30-3 genes were more frequent in patients with an obvious infection-related episode at onset, and correlated to expression levels of interferon response genes in plasmablasts. Collectively, these results imply that B cell responses in ME/CFS are directed against an infectious agents or priming antigens induced before disease onset.

Source: Sato W, Ono H, Matsutani T, Nakamura M, Shin I, Amano K, Suzuki R, Yamamura T. Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome. Brain Behav Immun. 2021 Mar 29:S0889-1591(21)00153-7. doi: 10.1016/j.bbi.2021.03.023. Epub ahead of print. PMID: 33794313. https://pubmed.ncbi.nlm.nih.gov/33794313/

Recursive ensemble feature selection provides a robust mRNA expression signature for myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disorder characterized by disabling fatigue. Several studies have sought to identify diagnostic biomarkers, with varying results. Here, we innovate this process by combining both mRNA expression and DNA methylation data. We performed recursive ensemble feature selection (REFS) on publicly available mRNA expression data in peripheral blood mononuclear cells (PBMCs) of 93 ME/CFS patients and 25 healthy controls, and found a signature of 23 genes capable of distinguishing cases and controls.

REFS highly outperformed other methods, with an AUC of 0.92. We validated the results on a different platform (AUC of 0.95) and in DNA methylation data obtained from four public studies on ME/CFS (99 patients and 50 controls), identifying 48 gene-associated CpGs that predicted disease status as well (AUC of 0.97). Finally, ten of the 23 genes could be interpreted in the context of the derailed immune system of ME/CFS.

Source: Metselaar, P.I., Mendoza-Maldonado, L., Li Yim, A.Y.F. et al. Recursive ensemble feature selection provides a robust mRNA expression signature for myalgic encephalomyelitis/chronic fatigue syndrome. Sci Rep 11, 4541 (2021). https://doi.org/10.1038/s41598-021-83660-9 https://www.nature.com/articles/s41598-021-83660-9 (Full text)

A Molecular network approach reveals shared cellular and molecular signatures between chronic fatigue syndrome and other fatiguing illnesses

Abstract:

The molecular mechanisms of chronic fatigue syndrome (CFS, or Myalgic encephalomyelitis), a disease defined by extreme, long-term fatigue, remain largely uncharacterized, and presently no molecular diagnostic test and no specific treatments exist to diagnose and treat CFS patients. While CFS has historically had an estimated prevalence of 0.1-0.5% [1], concerns of a “long hauler” version of Coronavirus disease 2019 (COVID-19) that symptomatically overlaps CFS to a significant degree (Supplemental Table-1) and appears to occur in 10% of COVID-19 patients[2], has raised concerns of a larger spike in CFS [3].

Here, we established molecular signatures of CFS and a corresponding network-based disease context from RNA-sequencing data generated on whole blood and FACs sorted specific peripheral blood mononuclear cells (PBMCs) isolated from CFS cases and non-CFS controls. The immune cell type specific molecular signatures of CFS we identified, overlapped molecular signatures from other fatiguing illnesses, demonstrating a common molecular etiology. Further, after constructing a probabilistic causal model of the CFS gene expression data, we identified master regulator genes modulating network states associated with CFS, suggesting potential therapeutic targets for CFS.

Source: Comella PH, Gonzalez-Kozlova E, Kosoy R, Charney AW, Peradejordi IF, Chandrasekar S, Tyler SR, Wang W, Losic B, Zhu J, Hoffman GE, Kim-Schulze S, Qi J, Patel M, Kasarskis A, Suarez-Farinas M, Gümüş ZH, Argmann C, Merad M, Becker C, Beckmann ND, Schadt EE. A Molecular network approach reveals shared cellular and molecular signatures between chronic fatigue syndrome and other fatiguing illnesses. medRxiv [Preprint]. 2021 Feb 2:2021.01.29.21250755. doi: 10.1101/2021.01.29.21250755. PMID: 33564792; PMCID: PMC7872387.  https://pubmed.ncbi.nlm.nih.gov/33564792/

Are Circulating FGF21 and NT-proBNP promising novel biomarkers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome?

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, disabling, and complex multisystem illness of unknown etiology. The protein FGF21 regulates glucose homeostasis and lipid metabolism, and the protein NT-proBNP is strongly associated with an elevated cardiovascular risk; however, little is known about their role in ME/CFS patients. To address this gap, we explored the association between FGF21 and NT-proBNP and oxidative stress and inflammatory markers in ME/CFS.

Twenty-one ME/CFS patients and 20 matched healthy controls were included in the study. Participants filled out validated self-reported questionnaires on their current health status covering demographic and clinical characteristics. Plasma showed significantly decreased total antioxidant capacity and increased lipoperoxides levels (p = 0.009 and p = 0.021, respectively) in ME/CFS. These ME/CFS patients also had significantly increased levels of inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α, and C-reactive protein (p < 0.05 for all) but not for IL-8 (p = 0.833) in ME/CFS, indicating low-grade systemic inflammation status. Circulating FGF21 and NT-proBNP levels were significantly higher (p < 0.0001 and p = 0.005, respectively) in ME/CFS patients than in healthy controls.

Significantly positive correlations were found between NT-proBNP levels and IL-1β and IL-6 (p = 0.04 and p = 0.01) in ME/CFS patients but not between FGF21 and these cytokines. In contrast, no significant correlations were found for either FGF21 or NT-proBNP in controls. These findings lead to the hypothesis that elevated FGF21 and NT-proBNP levels and the association between NT-proBNP and inflammation may be promising novel diagnostic and therapeutic targets in ME/CFS.

Source: Domingo JC, Cordobilla B, Ferrer R, Giralt M, Alegre-Martin J, Castro-Marrero J. Are Circulating FGF21 and NT-proBNP promising novel biomarkers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome? Antioxid Redox Signal. 2020 Dec 22. doi: 10.1089/ars.2020.8230. Epub ahead of print. PMID: 33353469. https://pubmed.ncbi.nlm.nih.gov/33353469/

Risks for Developing ME/CFS in College Students Following Infectious Mononucleosis: A Prospective Cohort Study

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) involves severe fatigue, unrefreshing sleep, and cognitive impairment, leading to functional difficulties; prior studies have not evaluated risk factors with behavioral and immune data collected prior to developing ME/CFS.. Up to 5% of university students develop infectious mononucleosis (IM) annually, and 9-12% meet criteria for ME/CFS six months later. We sought to determine predictors of ME/CFS.

Methods: We enrolled college students at the start of the school year (Time 1), identified those who developed IM (Time 2) and followed them for 6 months (Time 3), identifying three groups: those who developed ME/CFS, those who developed severe ME/CFS (meeting >1 set of criteria) and those who were asymptomatic. We conducted 8 behavioral and psychological surveys and analyzed cytokines at three time points.

Results: 238 of the 4501 students (5.3%) developed IM; 6 months later, 55 of the 238 (23%) met criteria for ME/CFS and 157 (66%) were asymptomatic. 67 of the 157 asymptomatic students served as controls. Students with severe-ME/CFS were compared to students who were asymptomatic at three time points. The former group was not different from the latter group at Time 1 (prior to developing IM) in stress, coping, anxiety or depression, but were different in several behavioral measures and had significantly lower levels of IL-6 and IL-13. At Time 2 (when they developed IM), the two ME/CFS groups tended to have more autonomic complaints and behavioral symptoms while the severe- ME/CFS group had higher levels of IL-12 and lower levels of IL-13 than the recovered group.

Conclusion: At baseline, those who developed ME/CFS had more physical symptoms and immune irregularities, but not more psychological symptoms, than those who recovered.

Source: Leonard A Jason, PhD, Joseph Cotler, PhD, Mohammed F Islam, PhD, Madison Sunnquist, PhD, Ben Z Katz, MD, Risks for Developing ME/CFS in College Students Following Infectious Mononucleosis: A Prospective Cohort Study, Clinical Infectious Diseases, , ciaa1886, https://doi.org/10.1093/cid/ciaa1886

The antinuclear antibody dense fine speckled pattern and possible clinical associations: An indication of a proinflammatory microenvironment

Abstract:

Background: Indirect immunofluorescence (IIF) is the most prevalent screening antinuclear antibody test for systemic autoimmune rheumatic disease (SARD). Certain IIF patterns have known antibody and disease associations, but the dense fine speckled (ANA-DFS) pattern has no confirmed clinical associations. Our objective was to determine the prevalence of SARD among a group of ANA-DFS positive individuals and to identify final diagnoses among non-SARD individuals in order to determine possible clinical associations with the ANA-DFS pattern.

Methods: A retrospective study of 425 patients from a university health care system with a positive ANA-DFS pattern consecutively between August 2017 and September 2018. Sera samples underwent ANA testing by IIF on HEp-2 cell substrates (Euroimmun, Germany). Clinical information was retrieved from electronic health records and stored in a de-identified database.

Results: The prevalence of SARD was 24%. Undetermined diagnosis (17%), skin disorders (12.1%), and fibromyalgia/chronic pain syndrome/chronic fatigue syndrome (11.8%) were the most common non-SARD diagnoses. Taking into account past medical history, the most common non-SARD were atopic disorders (21.2%), fibromyalgia/chronic pain syndrome/chronic fatigue syndrome (17.6%), and skin disorders (16.7%).

Conclusions: The ANA-DFS pattern may be indicative of an underlying antigen-antibody interaction that plays a role in either the initiation or propagation of immunologic reactions. DFS70/LEDGF is a transcription factor involved in cell survival and stress protection, and autoantibodies may inhibit its function. It is likely that there are other antibodies producing the ANA-DFS pattern besides anti-DFS70/LEDGF, and more research is necessary to identify additional antibody specificities. The ANA-DFS pattern may be an indicator of a proinflammatory microenvironment given the high frequency of symptomatic patients and disease processes with an immunologic basis (including SARD).

Source: Lundgren MC, Sapkota S, Peterson DJ, Crosson JT. The antinuclear antibody dense fine speckled pattern and possible clinical associations: An indication of a proinflammatory microenvironment. J Immunol Methods. 2020 Oct 26:112904. doi: 10.1016/j.jim.2020.112904. Epub ahead of print. PMID: 33121975. https://pubmed.ncbi.nlm.nih.gov/33121975/

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study

Abstract:

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls.

Methods: We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed.

Results: ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association.

Conclusions: Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

Source: Giloteaux L, O’Neal A, Castro-Marrero J, Levine SM, Hanson MR. Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study. J Transl Med. 2020 Oct 12;18(1):387. doi: 10.1186/s12967-020-02560-0. PMID: 33046133. https://pubmed.ncbi.nlm.nih.gov/33046133/

Autoantibodies to beta-adrenergic and muscarinic cholinergic receptors in Myalgic Encephalomyelitis (ME) patients – A validation study in plasma and cerebrospinal fluid from two Swedish cohorts

Abstract:

Myalgic encephalomyelitis (ME) also known as ME/CFS (Chronic Fatigue Syndrome) or ME/SEID (Systemic Exertion Intolerance Disorder), is a disabling and often long-lasting disease that can drastically impair quality of life and physical/social functioning of the patients. Underlying pathological mechanisms are to a large extent unknown, but the presence of autoantibodies, cytokine pattern deviations and the presentation of cognitive and autonomic nervous system related symptoms provide evidence for ME being an immunological disorder with elements of autoimmunity. Increased levels of autoantibodies binding to adrenergic and muscarinic receptors in ME-patients have been reported. It is hypothesized that these autoantibodies have pathological significance and contribute to the ME-specific symptoms, however, these observations need to be validated.

This study was designed to investigate potential differences in adrenergic and muscarinic receptor autoantibody levels in plasma and cerebrospinal fluid (CSF) samples between ME patients and gender and age-matched healthy controls, and to correlate the autoantibody levels to disease severity.

We collected bodyfluids and health-related questionnaires from two Swedish ME cohorts, plasma and CSF from one of the cohorts (n ​= ​24), only plasma from the second cohort (n ​= ​24) together with plasma samples (n ​= ​24) and CSF (n ​= ​6) from healthy controls.

All samples were analysed for IgG autoantibodies directed against Alpha- (α1, α2) and Beta- (β1-3) adrenergic receptors and Muscarinic (M) 1–5 acetylcholine receptors using an ELISA technique. The questionnaires were used as measures of disease severity.

Significant increases in autoantibody levels in ME patients compared to controls were found for M3 and M4 -receptors in both cohorts and β1, β2, M3 and M4-receptors in one cohort. No significant correlations were found between autoantibody levels and disease severity. No significant levels of autoantibodies were detected in the CSF samples. These findings support previous findings that there exists a general pattern of increased antibody levels to adrenergic and muscarinic receptors within the ME patient group. However, the role of increased adrenergic and muscarinic receptor autoantibodies in the pathogenesis of ME is still uncertain and further research is needed to evaluate the clinical significance of these findings.

Source: Annie Bynke; Per Julin; Carl-Gerhard Gottfries; Harald Heidecke; Carmen Scheibenbogen; Jonas Bergquist. Autoantibodies to beta-adrenergic and muscarinic cholinergic receptors in Myalgic Encephalomyelitis (ME) patients – A validation study in plasma and cerebrospinal fluid from two Swedish cohorts. Brain, Behavior, & Immunity – Health, ISSN: 2666-3546, Vol: 7, Page: 100107 https://www.sciencedirect.com/science/article/pii/S2666354620300727 (Full text)