Autoimmunity-Related Risk Variants in PTPN22 and CTLA4 Are Associated With ME/CFS With Infectious Onset

Single nucleotide polymorphisms (SNP) in various genes have been described to be associated with susceptibility to autoimmune disease. In this study, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients and controls were genotyped for five immune gene SNPs in tyrosine phosphatase non-receptor type 22 (PTPN22, rs2476601), cytotoxic T-lymphocyte-associated protein 4 (CTLA4, rs3087243), tumor necrosis factor (TNF, rs1800629 and rs1799724), and interferon regulatory factor 5 (IRF5, rs3807306), which are among the most important risk variants for autoimmune diseases. Analysis of 305 ME/CFS patients and 201 healthy controls showed significant associations of the PTPN22 rs2476601 and CTLA4 rs3087243 autoimmunity-risk alleles with ME/CFS. The associations were only found in ME/CFS patients, who reported an acute onset of disease with an infection (PTPN22 rs2476601: OR 1.63, CI 1.04-2.55, p = 0.016; CTLA4 rs3087243: OR 1.53, CI 1.17-2.03, p = 0.001), but not in ME/CFS patients without infection-triggered onset (PTPN22 rs2476601: OR 1.09, CI 0.56-2.14, p = 0.398; CTLA4 rs3087243: OR 0.89, CI 0.61-1.30, p = 0.268). This finding provides evidence that autoimmunity might play a role in ME/CFS with an infection-triggered onset. Both genes play a key role in regulating B and T cell activation

Charakterisierung der Immunmodulation durch den Transkriptionsfaktor FOXO3

Forkhead box O3 (FOXO3) is a transcription factor involved in cell metabolism, survival, longevity and inflammatory diseases. In the current thesis, we provided evidence that deletion of Foxo3 in mice enhances natural killer (NK) cell development, activity, cytotoxic function and IFNgamma (IFN-γ) expression leading to a better viral clearance in Coxsackievirus B3 (CVB3) infected hearts 3 days after infection and a lower inflammatory destruction of the heart tissue 7 days after infection. In line with this, we described an association of the gain-of-function single nucleotide polymorphism (SNP) rs12212067 with reduced IFN-γ production in NK cells. Metformin comprehensive pathway and functional analysis provided evidence that metformin activates FOXO3 via AMP-activated protein kinase (AMPK). Further, reactive oxygen and nitrogen species (ROS/RNS) production was reduced in human immune cells accompanied by an induction of gene expression of antioxidative enzymes superoxide dismutase 2 (SOD2) and cytochrome c (CYCS) being FOXO3 targets. This effect may be beneficial in chronic inflammation and atherosclerosis and may play a role in the improved morbidity and mortality of diabetes patients taking metformin. Ongoing trials (TAME, MILES) analyze the effect of metformin on morbidity and mortality in healthy older people. Based on our findings it would be very interesting to study the effect of metformin on FOXO3 activation in clinical trials and the potential influence of FOXO3 SNPs.Der Transkriptionsfaktor Forkhead-Box-Protein O3 (FOXO3) ist essentiell für die Regulation zahlreiche zelluläre Prozesse wie dem Metabolismus, der Proliferation und der Apoptose. Des Weiteren ist FOXO3 mit Langlebigkeit assoziiert und spielt eine Rolle bei der Immunzellregulation bei entzündlichen Erkrankungen. Die Ergebnisse dieser Arbeit zeigten, dass der Verlust von Foxo3 in der Maus die Entwicklung natürliche Killerzellen (NK Zellen) fördert und deren Aktivität, Zytotoxizität und Interferon-gamma (IFN-γ) Expression verstärkt. Dies führte zu einer verbesserten viralen Eliminierung in Coxsackievirus B3 (CVB3) infizierten Herzen und folglich zu einer geringeren entzündlich bedingten Schädigung des Herzgewebes. In humanen NK Zellen zeigte sich zudem, dass die expressionssteigernde Variante des Einzelnukleotid-Polymorphismus (SNP) rs12212067 im FOXO3 Gen mit einer geringeren IFN-γ Produktion assoziiert ist. Umfangreiche Untersuchungen von induzierten Signalwegen und Funktionen in humanen Immunzellen zeigten weiter, dass das Antidiabetikum Metformin FOXO3 abhängig von der AMP-aktivierte Proteinkinase (AMPK) aktiviert. Die FOXO3 Aktivierung führte weiter zur Induktion antioxidativ wirkender FOXO3 Zielgene: der mitochondrialen Superoxide dismutase 2 (SOD2) und des Cytochrom c (CYCS) und folglich zu einer Abnahme reaktiver Sauerstoff- und Stickstoffspezies (ROS/RNS). Eine Aktivierung von FOXO3 durch Metformin in Immunzellen kann bei chronischen Entzündungen sowie Atherosklerose vorteilhaft sein und auch die Verbesserung der Morbidität sowie Mortalität bei Patienten mit Diabetes unter Metformin-Therapie erklären. Laufende Studien (TAME, MILES) untersuchen zurzeit die Wirkung von Metformin auf die Morbidität und Mortalität bei älteren Menschen. Auf Grundlage der vorliegenden Arbeit wäre hierbei eine erweiterte Untersuchung der Bedeutung von FOXO3 Aktivierung unter Metformin-Therapie und der potentielle Einfluss von FOXO3 SNPs von weitreichendem Interesse.DFG, 264558061, Characterization of Cardioprotection and Immunmodulation by FOXO3a as a Master Regulator of Adiponecti

Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors.

Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff

Metformin Attenuates ROS via FOXO3 Activation in Immune Cells

Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067, rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient (Foxo3(-/-) ) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3(-/-) mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk

Peripheral endothelial dysfunction in myalgic encephalomyelitis/chronic fatigue syndrome

Aims Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystem disease. Evidence for disturbed vascular regulation comes from various studies showing cerebral hypoperfusion and orthostatic intolerance. The peripheral endothelial dysfunction (ED) has not been sufficiently investigated in patients with ME/CFS. The aim of the present study was to examine peripheral endothelial function in patients with ME/CFS. Methods and results Thirty-five patients [median age 40 (range 18–70) years, mean body mass index 23.8 ± 4.2 kg/m2, 31% male] with ME/CFS were studied for peripheral endothelial function assessed by peripheral arterial tonometry (EndoPAT2000). Clinical diagnosis of ME/CFS was based on Canadian Criteria. Nine of these patients with elevated antibodies against β2-adrenergic receptor underwent immunoadsorption, and endothelial function was measured at baseline and 3, 6, and 12 months follow-up. ED was defined by reactive hyperaemia index ≤1.81. Twenty healthy subjects of similar age and body mass index were used as a control group. Peripheral ED was found in 18 of 35 patients (51%) with ME/CFS and in 4 healthy subjects (20%, P < 0.05). Patients with ED, in contrast to patients with normal endothelial function, reported more severe disease according to Bell score (31 ± 12 vs. 40 ± 16, P = 0.04), as well as more severe fatigue-related symptoms (8.62 ± 0.87 vs. 7.75 ± 1.40, P = 0.04) including a higher demand for breaks [9.0 (interquartile range 7.0–10.0) vs. 7.5 (interquartile range 6.0–9.25), P = 0.04]. Peripheral ED showed correlations with more severe immune-associated symptoms (r = −0.41, P = 0.026), such as sore throat (r = −0.38, P = 0.038) and painful lymph nodes (r = −0.37, P = 0.042), as well as more severe disease according to Bell score (r = 0.41, P = 0.008) and symptom score (r = −0.59, P = 0.005). There were no differences between the patient group with ED and the patient group with normal endothelial function regarding demographic, metabolic, and laboratory parameters. Further, there was no difference in soluble vascular cell adhesion molecule and soluble intercellular adhesion molecule levels. At baseline, peripheral ED was observed in six patients who underwent immunoadsorption. After 12 months, endothelial function had improved in five of these six patients (reactive hyperaemia index 1.58 ± 0.15 vs. 2.02 ± 0.46, P = 0.06). Conclusions Peripheral ED is frequent in patients with ME/CFS and associated with disease severity and severity of immune symptoms. As ED is a risk factor for cardiovascular disease, it is important to elucidate if peripheral ED is associated with increased cardiovascular morbidity and mortality in ME/CFS

IgG stimulated β2 adrenergic receptor activation is attenuated in patients with ME/CFS

Background: There is emerging evidence of a network of natural autoantibodies against GPCR which is dysregulated in various diseases. β2 adrenergic and M3 and M4 cholinergic receptor (β2 AdR and M3/4 mAChR) antibodies were found to be elevated in a subset of ME/CFS patients. Methods: We comparatively analyzed the effects of polyclonal IgG on β2 AdR signaling and immune cell function in vitro. 16 IgG fractions were isolated from serum of 5 ME/CFS patients with elevated (CFS AABhigh) and 5 with normal levels (CFS AABnorm) of β2 AdR autoantibodies, and from 6 healthy controls (HC). The effect of each IgG on β-arrestin recruitment and cAMP production in β2 AdR and M3/4R reporter cell lines was studied. Further effect of each IgG on human monocyte cytokine production and on T cell proliferation in vitro was analyzed. In addition, studies on cytokine production in β2 AdR wild type and knockout mice splenocytes incubated with IgG fractions were performed. Results: We found that IgGs from HC could stimulate β-arrestin recruitment and cAMP production in β2 AdR reporter cell lines whereas IgGs from CFS AABhigh had no effect. The IgG-mediated activation of β2 AdR was confirmed in β2 AdR wt and ko mice. In accordance with previous studies IgG fractions from HC inhibited LPS-induced TNFα and stimulated LPS-induced IL-10 production of monocytes. Further IgG fractions from HC enhanced proliferation of T-cells stimulated with anti-CD3/CD28. IgG fractions from CFS AABhigh patients had no significant effect on both cytokine production and T cell proliferation, while IgGs from CFS AABnorm had an intermediate effect. We could also observe that IgG can modulate the signaling of β2 AdR ligands isoprenline and propranolol. Conclusions: We provide evidence that IgG can activate β2 AdR. The β2 AdR activation by IgG is attenuated in ME/CFS patients. A dysregulation of β2 AdR function could explain many symptoms of ME/CFS

Tolerability and Efficacy of s.c. IgG Self-Treatment in ME/CFS Patients with IgG/IgG Subclass Deficiency: A Proof-of-Concept Study

Background: Chronic fatigue syndrome (ME/CFS) is a complex disease frequently triggered by infections. IgG substitution may have therapeutic effect both by ameliorating susceptibility to infections and due to immunomodulatory effects. Methods: We conducted a proof of concept open trial with s.c. IgG in 17 ME/CFS patients suffering from recurrent infections and mild IgG or IgG subclass deficiency to assess tolerability and efficacy. Patients received s.c. IgG therapy of 0.8 g/kg/month for 12 months with an initial 2 months dose escalation phase of 0.2 g and 0.4 g/kg/month. Results: Primary outcome was improvement of fatigue assessed by Chalder Fatigue Scale (CFQ; decrease ≥ 6 points) and of physical functioning assessed by SF-36 (increase ≥ 25 points) at month 12. Of 12 patients receiving treatment per protocol 5 had a clinical response at month 12. Two additional patients had an improvement according to this definition at months 6 and 9. In four patients treatment was ceased due to adverse events and in one patient due to disease worsening. We identified LDH and soluble IL-2 receptor as potential biomarker for response. Conclusion: Our data indicate that self-administered s.c. IgG treatment is feasible and led to clinical improvement in a subset of ME/CFS patients

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Efficacy of Repeat Immunoadsorption

(1) Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmunological disease. There is evidence for an autoimmune mechanism for ME/CFS with an infection-triggered onset and dysfunction of &szlig;2-adrenoreceptor antibodies (&szlig;2AR-AB). In a first proof-of-concept study, we could show that IA was effective to reduce &szlig;2AR-AB and led to improvement of various symptoms. (2) Five of the ME/CFS patients who had clinical improvement following treatment with a five-day IA were retreated in the current study about two years later with a modified IA protocol. The severity of symptoms was assessed by disease specific scores during a follow-up period of 12 months. The antibodies were determined by ELISA. (3) The modified IA treatment protocol resulted in a remarkable similar clinical response. The treatment was well tolerated and 80&ndash;90% decline of total IgG and &szlig;2AR-AB was achieved. Four patients showed a rapid improvement in several clinical symptoms during IA therapy, lasting for six to 12 months. One patient had no improvement. (4) We could provide further evidence that IA has clinical efficacy in patients with ME/CFS. Data from our pilot trial warrant further controlled studies in ME/CFS

Demographic data of the donor groups.

<p>Demographic data of the donor groups.</p