Plasma mtDNA as a possible contributor to and biomarker of inflammation in rheumatoid arthritis.

OBJECTIVES: Neutrophil extracellular trap formation and cell-free DNA (cfDNA) contribute to the inflammation in rheumatoid arthritis (RA), but it is unknown if mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) is more abundant in the circulation. It is unclear if DNA concentration measurements may assist in clinical decision-making. METHODS: This single-center prospective observational study collected plasma from consecutive RA patients and healthy blood donors. Platelets were removed, and mtDNA and nDNA copy numbers were quantified by polymerase chain reaction (PCR). RESULTS: One hundred six RA patients and 85 healthy controls (HC) were recruited. Circulating median mtDNA copy numbers were increased 19.4-fold in the plasma of patients with RA (median 1.1 x108 copies/mL) compared to HC (median 5.4 x106 copies/mL, p<0.0001). Receiver operating characteristics (ROC) curve analysis of mtDNA copy numbers identified RA patients with high sensitivity (92.5%) and specificity (89.4%) with an area under the curve (AUC) of 0.97, p <0.0001 and a positive likelihood ratio of 8.7. Demographic, serological (rheumatoid factor (RF) positivity, anti-citrullinated protein antibodies (ACPA) positivity) and treatment factors were not associated with DNA concentrations. mtDNA plasma concentrations, however, correlated significantly with disease activity score-28- erythrocyte sedimentation rate (DAS28-ESR) and increased numerically with increasing DAS28-ESR and clinical disease activity index (CDAI) activity. MtDNA copy numbers also discriminated RA in remission (DAS28 <2.6) from HC (p<0.0001). Also, a correlation was observed between mtDNA and the ESR (p = 0.006, R= 0.29). Similar analyses showed no significance for nDNA. CONCLUSION: In contrast to nDNA, mtDNA is significantly elevated in the plasma of RA patients compared with HC. Regardless of RA activity, the abundance of circulating mtDNA is a sensitive discriminator between RA patients and HC. Further validation of the diagnostic value of mtDNA testing is required.

Primary prophylaxis with mTOR inhibitor enhances T cell effector function and prevents heart transplant rejection during talimogene laherparepvec therapy of squamous cell carcinoma.

The application of mammalian target of rapamycin inhibition (mTORi) as primary prophylactic therapy to optimize T cell effector function while preserving allograft tolerance remains challenging. Here, we present a comprehensive two-step therapeutic approach in a male patient with metastatic cutaneous squamous cell carcinoma and heart transplantation followed with concomitant longitudinal analysis of systemic immunologic changes. In the first step, calcineurin inhibitor/ mycophenolic acid is replaced by the mTORi everolimus to achieve an improved effector T cell status with increased cytotoxic activity (perforin, granzyme), enhanced proliferation (Ki67) and upregulated activation markers (CD38, CD69). In the second step, talimogene laherparepvec (T-VEC) injection further enhances effector function by switching CD4 and CD8 cells from central memory to effector memory profiles, enhancing Th1 responses, and boosting cytotoxic and proliferative activities. In addition, cytokine release (IL-6, IL-18, sCD25, CCL-2, CCL-4) is enhanced and the frequency of circulating regulatory T cells is increased. Notably, no histologic signs of allograft rejection are observed in consecutive end-myocardial biopsies. These findings provide valuable insights into the dynamics of T cell activation and differentiation and suggest that timely initiation of mTORi-based primary prophylaxis may provide a dual benefit of revitalizing T cell function while maintaining allograft tolerance.

Environmental signals perceived by the brain abate pro-metastatic monocytes by dampening glucocorticoids receptor signaling.

While positive social-behavioral factors predict longer survival in cancer patients, the underlying mechanisms are unknown. Since tumor metastasis are the major cancer mortality factor, we investigated how an enriched environment (EE) conductive to enhanced sensory, cognitive and motor stimulation impact metastatic progression in lungs following intravasation in the circulation. We find that mice housed in EE exhibited reduced number of lung metastatic foci compared to control mice housed in a standard environment (SE). Compared to SE mice, EE mice increased lung inflammation as early as 4 days after circulating tumor cells extravasation. The impact of environmental signals on lung metastasis is independent of adrenergic receptors signaling. By contrast, we find that serum corticosterone levels are lower in EE mice and that glucocorticoid receptor (GR) antagonist reduces the number of lung metastasis in SE mice. In addition, the difference of the number of lung metastasis between SE and EE mice is abolished when inflammatory monocytes are rendered deficient in GR signaling. This decreased GR signaling in inflammatory monocytes of SE mice results in an exacerbated inflammatory profile in the lung. Our study shows that not only EE reduces late stages of metastatic progression in lungs but disclose a novel anti-tumor mechanism whereby GR-dependent reprogramming of inflammatory monocytes can inhibit metastatic progression in lungs. Moreover, while inflammatory monocytes have been shown to promote cancer progression, they also have an anti-tumor effect, suggesting that their role is more complex than currently thought.

Mitochondrial DNA: a novel indicator of active inflammation in ANCA-associated vasculitides.

OBJECTIVES: ANCA-associated vasculitis (AAV) includes granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). ANCA triggers neutrophil extracellular trap formation, which releases either mitochondrial (mt) DNA or nuclear DNA (n) DNA, contributing to inflammation. Our aim was to prospectively examine the extent and nature of circulating DNA in AAV and the clinical utility of DNA quantification. METHODS: DNA was isolated from platelet-free plasma of consecutive GPA and MPA patients and healthy controls (HCs). mtDNA and nDNA copy numbers were quantified by PCR. Clinical data, including the BVAS, were collected. RESULTS: Ninety-two HCs (median age 51 years, 58.7% female) and 101 AAV patients (80 GPA, 21 MPA, median age 64 years, 50.5% female, BVAS range: 0-30) were included. Median mtDNA copies were 13-fold higher in patients with AAV than in HCs; nDNA concentrations did not differ. Patients with active AAV (BVAS > 0) had 4-fold higher median mtDNA copies than patients in remission (P = 0.03). mtDNA, unlike nDNA, correlated with BVAS (r = 0.30, P = 0.002) and was associated with AAV activity at multivariable analysis. Receiver operating characteristic curve analysis indicated that mtDNA quantification differentiates patients with active AAV (BVAS > 0) from HCs with 96.1% sensitivity and 98.9% specificity (area under the curve 0.99). In 27 AAV patients with follow-up, mtDNA changes but not CRP or ANCA-titres correlated with BVAS changes (r = 0.56, P = 0.002). CONCLUSIONS: mtDNA, unlike nDNA, is elevated in the plasma of AAV patients and may contribute to systemic inflammation. mtDNA could be superior to established biomarkers in the laboratory monitoring of AAV activity.

Identification of Serum Interleukin 6 Levels as a Disease Severity Biomarker in Facioscapulohumeral Muscular Dystrophy.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies in adults, displaying a progressive, frequently asymmetric involvement of a typical muscles' pattern. FSHD is associated with epigenetic derepression of the polymorphic D4Z4 repeat on chromosome 4q, leading to DUX4 retrogene toxic expression in skeletal muscles. Identifying biomarkers that correlate with disease severity would facilitate clinical management and assess potential FSHD therapeutics' efficacy. OBJECTIVES: This study purpose was to analyze serum cytokines to identify potential biomarkers in a large cohort of adult patients with FSHD. METHODS: We retrospectively measured the levels of 20 pro-inflammatory and regulatory cytokines in sera from 100 genetically confirmed adult FSHD1 patients. Associations between cytokine concentrations and various clinical scores were investigated. We then measured serum and muscle interleukin 6 (IL-6) levels in a validated FSHD-like mouse model, ranging in severity and DUX4 expression. RESULTS: IL-6 was identified as the only cytokine with a concentration correlating with several clinical severity and functional scores, including Clinical Severity Score, Manual Muscle Testing sum score, Brooke and Vignos scores. Further, FSHD patients displayed overall IL-6 levels more than twice high as control, and patients with milder phenotypes exhibited lower IL-6 serum concentration than those with severe muscular weakness. Lastly, an FSHD-like mouse model analysis confirmed that IL-6 levels positively correlate with disease severity and DUX4 expression. CONCLUSIONS: Serum IL-6, therefore, shows promise as a serum biomarker of FSHD severity in a large cohort of FSHD1 adult patients.

Circulating mitochondrial DNA copy numbers represent a sensitive marker for diagnosis and monitoring of disease activity in systemic lupus erythematosus.

OBJECTIVES: Cell-free DNA is involved in the pathogenesis of systemic lupus erythematosus (SLE) but the clinical value of cell-free DNA measurements in SLE is unknown. Our aim was therefore to examine the utility of mitochondrial (mt) DNA and nuclear (n) DNA quantification in SLE. METHODS: EDTA plasma was drawn from 103 consecutive patients with SLE and from 56 healthy blood donors. mtDNA and nDNA copy numbers were quantified by PCR from cell-free plasma. Clinical parameters were recorded prospectively. RESULTS: Circulating mtDNA copy numbers were increased 8.8-fold in the plasma of patients with SLE (median 6.6×107 /mL) compared with controls (median 7.6×106 /mL, p<0.0001). Among all 159 individuals, a cut-off set at 1.8×107 mtDNA copies in a receiver operated curve identified patients with SLE with 87.4% sensitivity and 94.6% specificity; the area under the curve was 0.95 (p<0.0001). mtDNA levels were independent of age or gender, but correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) on multivariable analysis (p=0.004). Conversely, SLEDAI was associated with prednisone dose (p<0.001), anti-double stranded DNA-titres (p=0.003) and mtDNA levels (p=0.005), but not nDNA copy numbers. In 33 patients with SLE with available follow-up, the changes of mtDNA, but not those of nDNA concentrations, robustly correlated with the evolution of the SLEDAI (r=0.55, p=0.001). CONCLUSIONS: Circulating mtDNA unlike nDNA molecules are markedly increased in SLE plasma. Regardless of disease activity, circulating mtDNA levels distinguish patients with SLE from healthy controls with high sensitivity and represent an independent marker of SLE activity.

Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity.

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

Blood cytokines differentiate bipolar disorder and major depressive disorder during a major depressive episode: Initial discovery and independent sample replication.

Bipolar disorder (BD) diagnosis currently relies on assessment of clinical symptoms, mainly retrospective and subject to memory bias. BD is often misdiagnosed as Major Depressive Disorder (MDD) resulting in ineffective treatment and worsened clinical outcome. The primary purpose of this study was to identify blood biomarkers that discriminate MDD from BD patients when in a depressed state. We have used clinical data and serum samples from two independent naturalistic cohorts of patients with a Major Depressive Episode (MDE) who fulfilled the criteria of either BD or MDD at inclusion. The discovery and replication cohorts consisted of 462 and 133 patients respectively. Patients were clinically assessed using standard diagnostic interviews, and clinical variables including current treatments were recorded. Blood was collected and serum assessed for levels of 31 cytokines using a sensitive multiplex assay. A penalized logistic regression model combined with nonparametric bootstrap was subsequently used to identify cytokines associated with BD. Interleukin (IL)-6, IL-10, IL-15, IL-27 and C-X-C ligand chemokine (CXCL)-10 were positively associated with BD in the discovery cohort. Of the five cytokines identified as discriminant features in the discovery cohort, IL-10, IL-15 and IL-27 were also positively associated with BD in the replication cohort therefore providing an external validation to our finding. Should our results be validated in a prospective cohort, they could provide new insights into the pathophysiological mechanisms of mood disorders.

Pancreatic nerve electrostimulation inhibits recent-onset autoimmune diabetes.

Vagus nerve stimulation can ameliorate autoimmune diseases such as rheumatoid arthritis by modulation of the immune system. Its efficacy for the treatment of type 1 diabetes has not been explored, in part because the nerves projecting to the pancreatic lymph nodes (pLNs) in mice are unmapped. Here, we map the nerve projecting to the pancreas and pLNs in mice and use a minimally invasive surgical procedure to implant micro-cuff electrodes onto the nerve. Pancreatic nerve electrical stimulation (PNES) resulted in ß-adrenergic receptor-mediated-accumulation of B and T cells in pLNs and reduced production of pro-inflammatory cytokines following lipopolysaccharide stimulation. Autoreactive T cells showed reduced proliferation in pLNs of mice receiving PNES as compared to sham controls. In a spontaneous mouse model of autoimmune diabetes, PNES inhibited disease progression in diabetic mice.

Differential effects of specific cathepsin S inhibition in biocompartments from patients with primary Sjögren syndrome.

OBJECTIVE: Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. METHODS: Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1ß, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. RESULTS: CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. CONCLUSION: CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.

Correction: Stratification and prediction of remission in first-episode psychosis patients: the OPTiMiSE cohort study.

The original Article did not feature the list of collaborators. This has now been corrected in the PDF and HTML versions of this Article.

Apical splenic nerve electrical stimulation discloses an anti-inflammatory pathway relying on adrenergic and nicotinic receptors in myeloid cells.

The autonomic nervous system innervates all lymphoid tissues including the spleen therefore providing a link between the central nervous system and the immune system. The only known mechanism of neural inhibition of inflammation in the spleen relies on the production of norepinephrine by splenic catecholaminergic fibers which binds to ß2-adrenergic receptors (ß 2-ARs) of CD4+ T cells. These CD4+ T cells trigger the release of acetylcholine that inhibits the secretion of inflammatory cytokines by macrophages through α7 nicotinic acetylcholine receptor (α7nAchRs) signaling. While the vagal anti-inflammatory pathway has been extensively studied in rodents, it remains to be determined whether it coexists with other neural pathways. Here, we have found that three nerve branches project to the spleen in mice. While two of these nerves are associated with an artery and contain catecholaminergic fibers, the third is located at the apex of the spleen and contain both catecholaminergic and cholinergic fibers. We found that electrical stimulation of the apical nerve, but not the arterial nerves, inhibited inflammation independently of lymphocytes. In striking contrast to the anti-inflammatory pathway mechanism described so far, we also found that the inhibition of inflammation by apical nerve electrical stimulation relied on signaling by both ß 2-ARs and α7nAchRs in myeloid cells, with these two signaling pathways acting in parallel. Most importantly, apical splenic nerve electrical stimulation mitigated clinical symptoms in a mouse model of rheumatoid arthritis further providing the proof-of-concept that such an approach could be beneficial in patients with Immune-mediated inflammatory diseases.

Stratification and prediction of remission in first-episode psychosis patients: the OPTiMiSE cohort study.

Early response to first-line antipsychotic treatments is strongly associated with positive long-term symptomatic and functional outcome in psychosis. Unfortunately, attempts to identify reliable predictors of treatment response in first-episode psychosis (FEP) patients have not yet been successful. One reason for this could be that FEP patients are highly heterogeneous in terms of symptom expression and underlying disease biological mechanisms, thereby impeding the identification of one-size-fits-all predictors of treatment response. We have used a clustering approach to stratify 325 FEP patients into four clinical subtypes, termed C1A, C1B, C2A and C2B, based on their symptoms assessed using the Positive and Negative Syndrome Scale (PANSS) scale. Compared to C1B, C2A and C2B patients, those from the C1A subtype exhibited the most severe symptoms and were the most at risk of being non-remitters when treated with the second-generation antipsychotic drug amisulpride. Before treatment, C1A patients exhibited higher serum levels of several pro-inflammatory cytokines and inflammation-associated biomarkers therefore validating our stratification approach on external biological measures. Most importantly, in the C1A subtype, but not others, lower serum levels of interleukin (IL)-15, higher serum levels of C-X-C motif chemokine 12 (CXCL12), previous exposure to cytomegalovirus (CMV), use of recreational drugs and being younger were all associated with higher odds of being non-remitters 4 weeks after treatment. The predictive value of this model was good (mean area under the curve (AUC) = 0.73 ± 0.10), and its specificity and sensitivity were 45 ± 0.09% and 83 ± 0.03%, respectively. Further validation and replication of these results in clinical trials would pave the way for the development of a blood-based assisted clinical decision support system in psychosis.

CD8+ T cells are essential for the effects of enriched environment on hippocampus-dependent behavior, hippocampal neurogenesis and synaptic plasticity.

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNFα release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.