Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBP beta, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kappa B (NF-kappa B) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation

Butyrylcholinesterase and Acetylcholinesterase polymorphisms in Multiple Sclerosis patients: Implication in peripheral inflammation

Multiple Sclerosis (MS) is an autoimmune disease, having not fully understood aetiology, and both genetic and environmental factors contribute to the pathogenesis of the disease. The cholinergic system has been indicated as a mediator of neuro-immune interactions, as well as an internal regulator of immune responses. The aim of the present research was to assess the associations between BChE and AChE genetic variations and serum cholinergic and inflammatory profiles in 102 Relapsing Remitting-MS patients and 117 healthy controls. An increased frequency of the BChE K-allele in MS patients as compared to controls was found. In addition, data showed that patients had higher BChE enzymatic activity, which is increased by the presence of the polymorphic allele and reduced amounts of circulating ACh. AChE polymorphism was significantly associated to reduced activity in both patients and controls. We propose that serum BChE and AChE activity may be used as a secondary markers to assess the role of non-neuronal cholinergic system in regulating peripheral inflammation via ACh regulation. This pilot study shed light on the role of the non-neuronal cholinergic system in immune cells to better understand MS pathogenesis. The cross-talk between the periphery and the CNS could have a new undescribed crucial role for MS, regarded as a systemic disease

Human neutrophils activated by TLR8 agonists, with or without IFN\u3b3, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct \u3b1 (IL-12A, IL-23A, and IL-27A) with two \u3b2 (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFN\u3b3 for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNF\u3b1 and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFN\u3b3 in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFN\u3b3, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFN\u3b3 plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL‐12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL‐12 family member subunits by RNA‐seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8‐mediated inducible expression of IL‐12B and IL‐23A, but not IL‐12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP‐seq), and subsequent production of IL‐23 and IL‐12B, but no IL‐12, proteins. Induction of IL‐23 requires endogenous TNF‐α, as both mRNA and protein levels were blocked in TLR8‐activated neutrophils via a TNF‐α‐neutralizing Ab. We also show that supernatants from TLR8‐activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL‐23‐dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL‐23, further supporting the key roles played by these cells in the important IL‐17/IL‐23 network and Th17 responses

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections

A multicenter study on the diagnostic significance of a single cerebrospinal fluid IgG band

The analysis of paired cerebrospinal fluid (CSF) and serum samples with isolectric focusing (IEF) can yield different patterns which can be of aid in the differential diagnosis of central nervous system (CNS) disorders. Rarely, a single CSF-restricted IgG band, which is not included within these patterns, can be detected in association with inflammatory disorders, multiple sclerosis (MS) above all. However, the diagnostic meaning of this abnormality is still uncertain. The main aim of our multicenter study was to establish the frequency and disease associations of single CSF IgG bands. Differences in the CSF profiles between MS and other diseases, and the follow-up patterns were also evaluated. Medical records of patients who underwent CSF analysis, which included IEF, over a 11.5-year period were retrospectively scrutinized at the participating centers, which used similar IEF techniques. One hundred and fifty-one of 9422 CSF reports (1.6%) showed single CSF-restricted IgG bands. Of the 129 patients with a definite diagnosis, 58.2% had CNS inflammatory-demyelinating diseases (the most frequent being MS: 21.7%), 6.2% tumours, 5.4% inflammatory peripheral nervous system diseases and 30.2% miscellaneous diseases. At follow-up, 3 out of the 10 patients with a repeated CSF analysis had developed an oligoclonal band pattern. Our findings indicate that single CSF IgG bands tend to associate with diseases characterized by the involvement of intrathecal humoral immune responses, and strongly support the notion that this abnormality should be regularly reported, thus alerting clinicians of possible inflammatory disorders of the CNS

Deoxyguanosine kinase deficiency: natural history and liver transplant outcome

Autosomal recessive pathogenetic variants in the DGUOK gene cause deficiency of deoxyguanosine kinase activity and mitochondrial deoxynucleotides pool imbalance, consequently, leading to quantitative and/or qualitative impairment of mitochondrial DNA synthesis. Typically, patients present early-onset liver failure with or without neurological involvement and a clinical course rapidly progressing to death. This is an international multicentre study aiming to provide a retrospective natural history of deoxyguanosine kinase deficient patients. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centres or clinicians all around the world caring for previously reported patients were contacted to provide followup information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of deoxyguanosine kinase deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from a systematic literature review, were analyzed. Patients had a neonatal onset (≤ 1 month) in 55.7% of cases, infantile (>1 month and ≤ 1 year) in 32.3%, pediatric (>1 year and ≤18 years) in 2.5% and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed statistically different survival rates (P < 0.0001) among the four age groups with the highest mortality for neonatal onset. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype–phenotype correlation was identified. Liver transplant significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after liver transplant. In conclusion, deoxyguanosine kinase deficiency is a disease spectrum with a prevalent liver and brain tissue specificity in neonatal and infantile-onset patients and muscle tissue specificity in adult-onset cases. Our study provides clinical, molecular genetics and biochemical data for early diagnosis, clinical trial planning and immediate intervention with liver transplant and/or nucleoside supplementation

Prognostic value of single-subject grey matter networks in early multiple sclerosis

The identification of prognostic markers in early multiple sclerosis (MS) is challenging and requires reliable measures that robustly predict future disease trajectories. Ideally, such measures should make inferences at the individual level to inform clinical decisions. This study investigated the prognostic value of longitudinal structural networks to predict five-year EDSS progression in patients with relapsing-remitting MS (RRMS). We hypothesized that network measures, derived from magnetic resonance imaging (MRI), outperform conventional MRI measurements at identifying patients at risk of developing disability progression. This longitudinal, multicentre study within the Magnetic Resonance Imaging in MS (MAGNIMS) network included 406 patients with RRMS (mean age = 35.7 ± 9.1 years) followed up for five years (mean follow-up = 5.0 ± 0.6 years). Expanded Disability Status Scale (EDSS) was determined to track disability accumulation. A group of 153 healthy subjects (mean age = 35.0 ± 10.1 years) with longitudinal MRI served as controls. All subjects underwent MRI at baseline and again one year after baseline. Grey matter (GM) atrophy over one year and white matter (WM) lesion load were determined. A single-subject brain network was reconstructed from T1-weighted scans based on GM atrophy measures derived from a statistical parameter mapping (SPM)-based segmentation pipeline. Key topological measures, including network degree, global efficiency and transitivity, were calculated at single-subject level to quantify network properties related to EDSS progression. Areas under receiver operator characteristic (ROC) curves were constructed for GM atrophy, WM lesion load and the network measures, and comparisons between ROC curves were conducted. The applied network analyses differentiated patients with RRMS who experience EDSS progression over five years through lower values for network degree [H(2)=30.0, p<0.001] and global efficiency [H(2)=31.3, p<0.001] from healthy controls but also from patients without progression. For transitivity, the comparisons showed no difference between the groups (H(2)= 1.5, p=0.474). Most notably, changes in network degree and global efficiency were detected independent of disease activity in the first year. The described network reorganization in patients experiencing EDSS progression was evident in the absence of GM atrophy. Network degree and global efficiency measurements demonstrated superiority of network measures in the ROC analyses over GM atrophy and WM lesion load in predicting EDSS worsening (all p-values < 0.05). Our findings provide evidence that GM network reorganization over one year discloses relevant information about subsequent clinical worsening in RRMS. Early GM restructuring towards lower network efficiency predicts disability accumulation and outperforms conventional MRI predictors