Levamisole Modulation of Podocytes' Actin Cytoskeleton in Nephrotic Syndrome.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Identification of cerebrospinal fluid biomarkers for parkinsonism using a proteomics approach.

The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson's disease (PD, n = 10), multiple system atrophy patients (MSA, n = 5) and non-neurological controls (n = 10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n = 46), atypical parkinsonism patients (AP; MSA, n = 17; Progressive supranuclear palsy; n = 8) and non-neurological controls (n = 39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that the discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease.

Background: The etiology of cerebral small vessel disease (SVD) remains elusive, though evidence is accumulating that inflammation contributes to its pathophysiology. We recently showed retrospectively that pro-inflammatory monocytes are associated with the long-term progression of white matter hyperintensities (WMHs). In this prospective high-frequency imaging study, we hypothesize that the incidence of SVD progression coincides with a pro-inflammatory monocyte phenotype. Methods: Individuals with SVD underwent monthly magnetic resonance imaging (MRI) for 10 consecutive months to detect SVD progression, defined as acute diffusion-weighted imaging-positive (DWI+) lesions, incident microbleeds, incident lacunes, and WMH progression. Circulating inflammatory markers were measured, cytokine production capacity of monocytes was assessed after ex vivo stimulation, and RNA sequencing was performed on isolated monocytes in a subset of participants. Results: 13 out of 35 individuals developed SVD progression (70 ± 6 years, 54% men) based on incident lesions (n = 7) and/or upper quartile WMH progression (n = 9). Circulating E-selectin concentration (p < 0.05) and the cytokine production capacity of interleukin (IL)-1ß and IL-6 (p < 0.01) were higher in individuals with SVD progression. Moreover, RNA sequencing revealed a pro-inflammatory monocyte signature including genes involved in myelination, blood-brain barrier, and endothelial-leukocyte interaction. Conclusions: Circulating monocytes of individuals with progressive SVD have an inflammatory phenotype, characterized by an increased cytokine production capacity and a pro-inflammatory transcriptional signature.

Autoantibody profiles in systemic sclerosis; a comparison of diagnostic tests.

OBJECTIVES: Autoimmune antibody profiling plays a prominent role in both classification and prognosis of systemic sclerosis (SSc). In the last years novel autoantibodies have been discovered and have become available in diagnostic assays. However, standardization in autoimmune serology is lacking, which may have a negative impact on the added value of autoantibodies in diagnosis and prognosis of SSc. In this paper we describe the comparison of commercially available diagnostic assays for the detection of SSc-associated autoantibodies and explored the coexistence of multiple SSc-associated autoantibodies within patients. METHODS: Serum samples of 347 patients from the Nijmegen Systemic Sclerosis Cohort were included in this study. All patients fulfilled the ACR/EULAR 2013 classification criteria for SSc and were classified as DcSSc or LcSSc according to the Leroy and Medsger criteria. All samples were evaluated on standard laboratory diagnostic tests for detection of SSc-specific autoantibodies CENPA and CENPB (ACA), Scl-70 (ATA), RNA Polymerase III (rp11/155) (ARA), and SSc-associated autoantibodies Fibrillarin, Th-To, PM-scl75, PM-Scl100, RNP68/A/C, Ku, NOR90, and PDGFR from suppliers EUROIMMUN, D-tek and Thermo Fisher Scientific. RESULTS: We found that 79% of the patients was positive for one or more of the SSc autoantibodies. Overall, a high agreement was observed between the diagnostic methods for the SSC-specific autoantibodies listed in the ACR/EULAR criteria (ATA, ACA, and ARA) (Cohen's kappa 0.53-0.97). However, a lower agreement was found for SSc-associated autoantibodies PM-Scl, and Ku, as well as for the SSc-specific autoantibodies fibrillarin and Th-To. Furthermore, the data revealed that the presence of ATA, ARA and ACA is predominantly mutually exclusive, with only a fraction of the patients testing positive for both ATA and ARA. CONCLUSION: Our data showed high concordance of prevalent SSc-specific autoantibodies between different diagnostic assays. Further standardisation for low prevalent SSc-specific and SSc-associated autoantibodies is needed.

Catecholamines Induce Trained Immunity in Monocytes In Vitro and In Vivo.

RATIONALE: Exposure to high catecholamine levels is associated with inflammatory changes of myeloid cells and atherosclerosis, but the underlying mechanisms are only partly understood. OBJECTIVE: To investigate whether the proinflammatory effects of noradrenaline and adrenaline can, in part, be explained by the induction of an immunologic memory in innate immune cells, termed trained immunity. METHODS AND RESULTS: In vitro, we exposed human primary monocytes to (nor)adrenaline for 24 hours, after which cells were rested and differentiated to macrophages over 5 days. After restimulation with lipopolysaccharide on day 6, (nor)adrenaline-exposed cells showed increased TNF-α (tumor necrosis factor-α) production. This coincided with an increase in glycolysis and oxidative phosphorylation measured with Seahorse technology on day 6 before restimulation. Inhibition of the ß-adrenoreceptor-cAMP signaling pathway prevented the induction of training. In vivo, we studied the functional, transcriptional, and epigenetic impact of peak-wise exposure to high catecholamine levels on monocytes isolated from pheochromocytoma/paraganglioma (PHEO) patients. In PHEO patients (n=10), the peripheral blood cell composition showed a myeloid bias and an increase of the inflammatory CD14++CD16+ (cluster of differentiation) intermediate monocyte subset compared with controls with essential hypertension (n=14). Ex vivo production of proinflammatory cytokines was higher in PHEO patients. These inflammatory changes persisted for 4 weeks after surgical removal of PHEO. Transcriptome analysis of circulating monocytes at baseline showed various differentially expressed genes in inflammatory pathways in PHEO patients; epigenetic profiling of the promoters of these genes suggests enrichment of the transcriptionally permissive chromatin mark H3K4me3 (trimethylation of lysine 4 on histone H3), indicative of in vivo training. CONCLUSIONS: Catecholamines induce long-lasting proinflammatory changes in monocytes in vitro and in vivo, indicating trained immunity. Our data contribute to the understanding of pathways driving inflammatory changes in conditions characterized by high catecholamine levels and propose that trained immunity underlies the increased cardiovascular event rate in PHEO patients.

Diagnostic profiles for precision medicine in systemic sclerosis; stepping forward from single biomarkers towards pathophysiological panels.

Systemic sclerosis (SSc) is an autoimmune disease which is characterized by vasculopathy, tissue fibrosis and activation of the innate and adaptive immune system. Clinical features of the disease consists of skin thickening and internal organ involvement. Due to the heterogeneous nature of the disease it is difficult to predict disease progression and complications. Despite the discovery of novel autoantibodies associated with SSc, there is an unmet need for biomarkers for diagnosis, disease progression and response to treatment. To date, the use of single (surrogate) biomarkers for these purposes has been unsuccessful. Combining multiple biomarkers in to predictive panels or ultimately algorithms could be more precise. Given the limited therapeutic options and poor prognosis of many SSc patients, a better understanding of the immune-pathofysiological profiles might aid to an adjusted therapeutic approach. Therefore, we set out to explore immunological fingerprints in various clinically defined forms of SSc. We used multilayer profiling to identify unique immune profiles underlying distinct autoantibody signatures. These immune profiles could fill the unmet need for prognosis and response to therapy in SSc. Here, we present 3 pathophysiological fingerprints in SSc based on the expression of circulating antibodies, vascular markers and immunomodulatory mediators.

Arterial Wall Inflammation and Increased Hematopoietic Activity in Patients With Primary Aldosteronism.

CONTEXT: Primary aldosteronism (PA) confers an increased risk of cardiovascular disease (CVD), independent of blood pressure. Animal models have shown that aldosterone accelerates atherosclerosis through proinflammatory changes in innate immune cells; human data are scarce. OBJECTIVE: The objective of this article is to explore whether patients with PA have increased arterial wall inflammation, systemic inflammation, and reprogramming of monocytes. DESIGN: A cross-sectional cohort study compared vascular inflammation on 2'-deoxy-2'-(18F)fluoro-D-glucose; (18F-FDG) positron emission tomography-computed tomography, systemic inflammation, and monocyte phenotypes and transcriptome between PA patients and controls. SETTING: This study took place at Radboudumc and Rijnstate Hospital, the Netherlands. PATIENTS: Fifteen patients with PA and 15 age-, sex-, and blood pressure-matched controls with essential hypertension (EHT) participated. MAIN OUTCOME MEASURES AND RESULTS: PA patients displayed a higher arterial 18F-FDG uptake in the descending and abdominal aorta (P < .01, P < .05) and carotid and iliac arteries (both P < .01). In addition, bone marrow uptake was higher in PA patients (P < .05). Although PA patients had a higher monocyte-to-lymphocyte ratio (P < .05), systemic inflammatory markers, cytokine production capacity, and transcriptome of circulating monocytes did not differ. Monocyte-derived macrophages from PA patients expressed more TNFA; monocyte-derived macrophages of healthy donors cultured in PA serum displayed increased interleukin-6 and tumor necrosis factor-α production. CONCLUSIONS: Because increased arterial wall inflammation is associated with accelerated atherogenesis and unstable plaques, this might importantly contribute to the increased CVD risk in PA patients. We did not observe inflammatory reprogramming of circulating monocytes. However, subtle inflammatory changes are present in the peripheral blood cell composition and monocyte transcriptome of PA patients, and in their monocyte-derived macrophages. Most likely, arterial inflammation in PA requires interaction between various cell types.