Inferring upstream regulatory genes of FOXP3 in human regulatory T cells from time-series transcriptomic data.

The discovery of upstream regulatory genes of a gene of interest still remains challenging. Here we applied a scalable computational method to unbiasedly predict candidate regulatory genes of critical transcription factors by searching the whole genome. We illustrated our approach with a case study on the master regulator FOXP3 of human primary regulatory T cells (Tregs). While target genes of FOXP3 have been identified, its upstream regulatory machinery still remains elusive. Our methodology selected five top-ranked candidates that were tested via proof-of-concept experiments. Following knockdown, three out of five candidates showed significant effects on the mRNA expression of FOXP3 across multiple donors. This provides insights into the regulatory mechanisms modulating FOXP3 transcriptional expression in Tregs. Overall, at the genome level this represents a high level of accuracy in predicting upstream regulatory genes of key genes of interest.

Early-to-mid stage idiopathic Parkinson's disease shows enhanced cytotoxicity and differentiation in CD8 T-cells in females.

Neuroinflammation in the brain contributes to the pathogenesis of Parkinson's disease (PD), but the potential dysregulation of peripheral immunity has not been systematically investigated for idiopathic PD (iPD). Here we showed an elevated peripheral cytotoxic immune milieu, with more terminally-differentiated effector memory (TEMRA) CD8 T, CD8+ NKT cells and circulating cytotoxic molecules in fresh blood of patients with early-to-mid iPD, especially females, after analyzing > 700 innate and adaptive immune features. This profile, also reflected by fewer CD8+FOXP3+ T cells, was confirmed in another subcohort. Co-expression between cytotoxic molecules was selectively enhanced in CD8 TEMRA and effector memory (TEM) cells. Single-cell RNA-sequencing analysis demonstrated the accelerated differentiation within CD8 compartments, enhanced cytotoxic pathways in CD8 TEMRA and TEM cells, while CD8 central memory (TCM) and naïve cells were already more-active and transcriptionally-reprogrammed. Our work provides a comprehensive map of dysregulated peripheral immunity in iPD, proposing candidates for early diagnosis and treatments.

Cardiac amyloidosis: a significant blind spot of the H2FPEF score.

BACKGROUND: Cardiac amyloidosis (CA) often mimics heart failure with preserved ejection fraction (HFpEF). Due to very different treatment strategies, an exact diagnosis and differentiation between pure HFpEF and CA-related heart failure (HF) is important. In the present study, we assessed the recently published H2FPEF score in patients with pure HFpEF, transthyretin (ATTR), as well as light chain (AL) amyloidosis-related HFpEF and tested whether it differentiates between these entities. METHODS: The H2FPEF scores consists of easy-to-assess clinical (Body Mass Index, number of hypertensive drugs, presence of atrial fibrillation, age) and echocardiographic (systolic pulmonary arterial pressure, E/E´) parameters. It can be computed in a categorical way resulting in scores between 0 and 9 points (0-1: HFpEF rule out, 2-5: further testing required, 6-9: HFpEF rule in), or in a continual way providing an exact percentage of a patient's HFpEF probability. Continuous and categorical variables were compared using the Kruskal-Wallis, Mann-Whitney-U, and χ2-tests. Diagnostic accuracy was computed from 2x2 tables. Survival analysis was performed with Kaplan-Meier curves. A P value of <0.05 was set as the level of significance. RESULTS: A total of 100 patients with pure HFpEF, 53 patients with ATTR, and 34 patients with AL CA were included in the present study. Median age (HFpEF: 71.5 years; ATTR CA: 77.0 years; AL CA: 60.0 years; P<0.001), gender distribution (HFpEF [female]: 73.0%, ATTR (female): 18.9%, AL [female]: 38.2%; P<0.001), and N-terminal prohormone of brain natriuretic peptide (HFpEF: 1045pg/mL; ATTR CA: 1927pg/mL; AL CA: 4308pg/mL; P<0.001) differed significantly between study cohorts. Median H2FPEF scores were highest among HFpEF (categorical: 5.0 points; continual: 95.1%), followed by ATTR (categorical: 4.0 points; continual: 89.0%), and AL CA (categorical: 3.0 points; continual: 31.2%). Respective P values were <0.001. Low H2FPEF scores (0-1 points) were found among patients in the AL CA cohort (29.4%), but not among HFpEF or ATTR CA patients (P<0.001). The majority of patients, irrespective of disease entity were in the intermediate score range (2-5 points, HFpEF: 80.0% ATTR CA: 94.3%, AL CA: 67.9%; P=0.006). High scores (6-9 points) were most often found among HFpEF patients (20.0%), followed by ATTR CA (5.7%) and AL CA (2.9%), (P=0.007). CONCLUSIONS: The H2FPEF score should be used with caution, as there is a significant overlap between HFpEF and CA-related HF.

Cardiopulmonary Long-Term Sequelae in Patients after Severe COVID-19 Disease.

We aimed to identify cardiopulmonary long-term effects after severe COVID-19 disease as well as predictors of Long-COVID in a prospective registry. A total of 150 consecutive, hospitalized patients (February 2020 and April 2021) were included six months post hospital discharge for a clinical follow-up. Among them, 49% experienced fatigue, 38% exertional dyspnea and 75% fulfilled criteria for Long-COVID. Echocardiography detected reduced global longitudinal strain (GLS) in 11% and diastolic dysfunction in 4%. Magnetic resonance imaging revealed traces of pericardial effusion in 18% and signs of former pericarditis or myocarditis in 4%. Pulmonary function was impaired in 11%. Chest computed tomography identified post-infectious residues in 22%. Whereas fatigue did not correlate with cardiopulmonary abnormalities, exertional dyspnea was associated with impaired pulmonary function (OR 3.6 [95% CI: 1.2-11], p = 0.026), reduced GLS (OR 5.2 [95% CI: 1.6-16.7], p = 0.003) and/or left ventricular diastolic dysfunction (OR 4.2 [95% CI: 1.03-17], p = 0.04). Predictors of Long-COVID included length of in-hospital stay (OR: 1.15 [95% CI: 1.05-1.26], p = 0.004), admission to intensive care unit (OR cannot be computed, p = 0.001) and higher NT-proBNP (OR: 1.5 [95% CI: 1.05-2.14], p = 0.026). Even 6 months after discharge, a majority fulfilled criteria for Long-COVID. While no associations between fatigue and cardiopulmonary abnormalities were found, exertional dyspnea correlated with impaired pulmonary function, reduced GLS and/or diastolic dysfunction.

Impact of Tafamidis and Optimal Background Treatment on Physical Performance in Patients With Transthyretin Amyloid Cardiomyopathy.

BACKGROUND: In patients with transthyretin amyloid cardiomyopathy, tafamidis was shown to slow the decline in 6-minute walking distance as compared with placebo. We aimed to define the impact of tafamidis and optimal background treatment on functional capacity as determined by cardiopulmonary exercise testing (CPET). METHODS: Seventy-eight consecutive patients were enrolled in the study. They underwent CPET at baseline, and outcome defined as death or heart failure hospitalization was obtained for a time period of up to 30 months. Fifty-four patients completed a follow-up CPET at 9±3 months (range, 4-16 months). Improvement in peak VO2 at follow-up was defined as ∆peak VO2≥1.0 mL/(kg·min), stable peak VO2 was defined as 0≤∆peak VO2<1.0 mL/(kg·min), and decline in peak VO2 was defined by ∆peak VO2<0 mL/(kg·min). RESULTS: Baseline peak VO2>14 mL/(kg·min) as well as minute ventilation/carbon dioxide production slope≤34 were associated with a lower risk of death or heart failure hospitalization (P=0.002, P=0.007, respectively). In 54 patients, who received tafamidis and underwent repeat CPET testing, an improvement in physical performance (P=0.002) was observed at follow-up. When comparing pre and post-treatment parameters, 29 patients (54%) showed an increase in percent predicted peak VO2 (P<0.0001), an improvement of peak VO2 (P<0.0001), and better physical performance at follow-up (P<0.0001). Patients with stable or improved peak VO2 had less advanced heart disease at baseline (P=0.046). CONCLUSIONS: Our findings demonstrate that baseline peak VO2 and baseline minute ventilation/carbon dioxide production slope predict outcomes and an improvement in physical performance as measured by CPET was observed in patients receiving tafamidis, who had less advanced disease at baseline, emphasizing the importance of early diagnosis.

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis during ageing.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4+ regulatory T cells (Treg cells). DJ-1 binds to PDHE1-ß (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs Treg survival starting in young mice and reduces Treg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible Treg cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As Treg homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish Treg homeostasis.

Combinatorial analysis reveals highly coordinated early-stage immune reactions that predict later antiviral immunity in mild COVID-19 patients.

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-ß levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients.

Stress hormone signalling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1.

Stress hormones are believed to skew the CD4 T-cell differentiation towards a Th2 response via a T-cell-extrinsic mechanism. Using isolated primary human naïve and memory CD4 T cells, here we show that both adrenergic- and glucocorticoid-mediated stress signalling pathways play a CD4 naïve T-cell-intrinsic role in regulating the Th1/Th2 differentiation balance. Both stress hormones reduced the Th1 programme and cytokine production by inhibiting mTORC1 signalling via two parallel mechanisms. Stress hormone signalling inhibited mTORC1 in naïve CD4 T cells (1) by affecting the PI3K/AKT pathway and (2) by regulating the expression of the circadian rhythm gene, period circadian regulator 1 (PER1). Both stress hormones induced the expression of PER1, which inhibited mTORC1 signalling, thus reducing Th1 differentiation. This previously unrecognized cell-autonomous mechanism connects stress hormone signalling with CD4 T-cell differentiation via mTORC1 and a specific circadian clock gene, namely PER1.

DJ-1 depletion prevents immunoaging in T-cell compartments.

Decline in immune function during aging increases susceptibility to different aging-related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T-cell subpopulations, still remain largely elusive. Here, we show that loss of DJ-1 encoded by PARK7/DJ-1, causing early-onset familial Parkinson's disease (PD), unexpectedly diminished signs of immunoaging in T-cell compartments of both human and mice. Compared with two gender-matched unaffected siblings of similar ages, the index PD patient with DJ-1 deficiency showed a decline in many critical immunoaging features, including almost doubled non-senescent T cells. The observation was further consolidated by the results in 45-week-old DJ-1 knockout mice. Our data demonstrated that DJ-1 regulates several immunoaging features via hematopoietic-intrinsic and naïve-CD8-intrinsic mechanisms. Mechanistically, DJ-1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T-cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ-1 in regulating immunoaging, discovering a potent target to interfere with immunoaging- and aging-associated diseases.

Standard Peripheral Blood Mononuclear Cell Cryopreservation Selectively Decreases Detection of Nine Clinically Relevant T Cell Markers.

Biobanking is an operational component of various epidemiological studies and clinical trials. Although peripheral blood is routinely acquired and stored in biobanks, the effects of specimen processing on cell composition and clinically relevant functional markers of T cells still require a systematic evaluation. In this study, we assessed 25 relevant T cell markers in human PBMCs and showed that the detection of nine membrane markers (e.g., PD-1, CTLA4, KLRG1, CD25, CD122, CD127, CCR7, and others reflecting exhaustion, senescence, and other functions) was reduced among at least one T cell subset following standard processing, although the frequency of CD4, CD8, and regulatory T cells was unaffected. Nevertheless, a 6-mo-long cryopreservation did not impair the percentages of cells expressing many other membrane and all the eight tested intracellular lineage or functional T cell markers. Our findings uncover that several clinically relevant markers are particularly affected by processing and the interpretation of those results in clinical trials and translational research should be done with caution.

Renin Feedback Is an Independent Predictor of Outcome in HFpEF.

Drugs which interact with the renin angiotensin aldosterone system (RAAS) aim to reduce the negative effects of angiotensin (Ang) II. Treatment with these drugs anticipate a compensatory up-regulation of renin; however, it has been shown that there is a large variability in circulating plasma renin (PRA), even in patients with optimal medical therapy in patients with heart failure (HF) with reduced ejection fraction (HFrEF). Our aim was to measure plasma renin activity (PRA-S), its response to RAAS inhibitor (RAASi) therapies and its effects on outcome in patients with HF with preserved ejection fraction (HFpEF). For this purpose, 150 HFpEF patients were included into a prospective single-center registry. Equilibrium (eq) angiotensin metabolites were measured from serum samples using mass spectroscopy. PRA-S (eqAng I + eqAng II) was calculated and compared in respect to the primary endpoint defined as all-cause death. PRA-S in patients with RAASi therapy was not significantly higher than in patients without RAASi (p = 0.262). Even after adjusting for confounding factors, PRA-S remained predictive for all-cause death in the multivariable model with a hazard ratio of 2.14 (95%CI 1.20-3.82, p = 0.010). We conclude that high PRA-S is associated with poor prognosis in patients with HFpEF, regardless of RAASi treatment, which could ultimately result in hyperactivated RAAS and consecutive negative effects on the cardiovascular and renal system, leading to poor outcome in patients with HFpEF.

Identification of VIMP as a gene inhibiting cytokine production in human CD4+ effector T cells.

Many players regulating the CD4+ T cell-mediated inflammatory response have already been identified. However, the critical nodes that constitute the regulatory and signaling networks underlying CD4 T cell responses are still missing. Using a correlation-network-guided approach, here we identified VIMP (VCP-interacting membrane protein), one of the 25 genes encoding selenoproteins in humans, as a gene regulating the effector functions of human CD4 T cells, especially production of several cytokines including IL2 and CSF2. We identified VIMP as an endogenous inhibitor of cytokine production in CD4 effector T cells via both the E2F5 transcription regulatory pathway and the Ca2+/NFATC2 signaling pathway. Our work not only indicates that VIMP might be a promising therapeutic target for various inflammation-associated diseases but also shows that our network-guided approach can significantly aid in predicting new functions of the genes of interest.

Prognostic implications of pericardial and pleural effusion in patients with cardiac amyloidosis.

BACKGROUND: Pericardial and pleural effusion are common findings in patients with cardiac amyloidosis (CA). It is not known, whether effusions correlate with right ventricular (RV) function in these patients. Furthermore, data on the prognostic significance of pleural and pericardial effusion in CA is scarce. METHODS: Patients with transthyretin (ATTR) and light chain (AL) CA were included in a clinical registry. All patients underwent transthoracic echocardiography at baseline. The presence of pericardial and pleural effusion was determined in every patient. The clinical endpoint was defined as cardiac death or heart failure hospitalization. RESULTS: In total, 143 patients were analysed. Of these, 85 patients were diagnosed with ATTR and 58 patients with AL. Twenty-four patients presented with isolated pericardial effusion and 35 with isolated pleural effusion. In 19 patients, both pericardial and pleural effusion were found and in 65 patients no effusion was present at baseline. The presence of pleural effusion correlated well with poor RV function, measured by global RV free-wall strain (p = 0.007) in patients with AL, but not in ATTR. No such correlation could be found for pericardial effusion in either amyloidosis subtype. Patients with AL presenting with pleural effusion had worse outcomes compared to patients with pericardial effusion alone or no effusion at baseline. In the ATTR group, there was no difference in outcomes according to presence and type of effusion. CONCLUSION: More than 50% of patients with CA presented with pleural and/or pericardial effusions. While pleural effusion was clearly associated with poor RV function in AL, we were not able to detect this association with pericardial effusion.

Mitochondria interaction networks show altered topological patterns in Parkinson's disease.

Mitochondrial dysfunction is linked to pathogenesis of Parkinson's disease (PD). However, individual mitochondria-based analyses do not show a uniform feature in PD patients. Since mitochondria interact with each other, we hypothesize that PD-related features might exist in topological patterns of mitochondria interaction networks (MINs). Here we show that MINs formed nonclassical scale-free supernetworks in colonic ganglia both from healthy controls and PD patients; however, altered network topological patterns were observed in PD patients. These patterns were highly correlated with PD clinical scores and a machine-learning approach based on the MIN features alone accurately distinguished between patients and controls with an area-under-curve value of 0.989. The MINs of midbrain dopaminergic neurons (mDANs) derived from several genetic PD patients also displayed specific changes. CRISPR/CAS9-based genome correction of alpha-synuclein point mutations reversed the changes in MINs of mDANs. Our organelle-interaction network analysis opens another critical dimension for a deeper characterization of various complex diseases with mitochondrial dysregulation.

Hemodynamic Profiles and Their Prognostic Relevance in Cardiac Amyloidosis.

This study sought to characterize cardiac amyloidosis (CA) patients with respect to hemodynamic parameters and asses their prognostic impact in different CA cohorts. Intracardiac and pulmonary arterial pressures (PAPs) are among the strongest predictors of outcomes in patients with heart failure (HF). Despite that, the hemodynamic profiles of patients with CA and their relation to prognosis have rarely been investigated. Invasive hemodynamic, clinical, and laboratory assessment, as well as cardiac magnetic resonance imaging were performed in our CA cohort. A total of 61 patients, 35 (57.4%) with wild-type transthyretin amyloidosis (ATTRwt) and 26 (42.6%) with light-chain amyloidosis (AL) were enrolled. ATTRwt patients had lower N-terminal prohormone of brain natriuretic peptide values and were less frequently in New York Heart Association class ≥ III. Intracardiac and PAPs were elevated, but hemodynamic parameters did not differ between CA groups. Whereas in ATTRwt, the median mean PAP (hazard ratio (HR): 1.130, p = 0.040) and pulmonary vascular resistance (HR: 1.010, p = 0.046) were independent predictors of outcome, no hemodynamic parameter was associated with outcome in the AL group. Cardiac ATTRwt and AL patients feature elevated intracardiac and PAPs and show similar hemodynamic profiles. However, hemodynamic parameters are of greater prognostic relevance in ATTRwt, potentially providing a new therapeutic target.

A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.