Citrulline enteral administration markedly reduces immunosuppressive extrafollicular plasma cell differentiation in a preclinical model of sepsis.

The sustained immunosuppression associated with severe sepsis favors an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations that expand during sepsis. In particular, sepsis supports CD39hi plasmablast metabolic reprogramming associated with adenosine-mediated suppressive activity. Arginine deficiency has been linked to an increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis-induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here, we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3+ and CD39+ plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC-mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in the splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses and plasma cell differentiation and further supports the development of citrulline-based clinical studies to prevent sepsis-associated immune dysfunction.

Multiple Sclerosis CSF Is Enriched With Follicular T Cells Displaying a Th1/Eomes Signature.

BACKGROUND AND OBJECTIVES: Tertiary lymphoid structures and aggregates are reported in the meninges of patients with multiple sclerosis (MS), especially at the progressive stage, and are strongly associated with cortical lesions and disability. Besides B cells, these structures comprise follicular helper T (Tfh) cells that are crucial to support B-cell differentiation. Tfh cells play a pivotal role in amplifying autoreactive B cells and promoting autoantibody production in several autoimmune diseases, but very few are known in MS. In this study, we examined the phenotype, frequency, and transcriptome of circulating cTfh cells in the blood and CSF of patients with relapsing-remitting MS (RRMS). METHODS: The phenotype and frequency of cTfh cells were analyzed in the blood of 39 healthy controls and 41 untreated patients with RRMS and in the CSF and paired blood of 10 patients with drug-naive RRMS at diagnosis by flow cytometry. Using an in vitro model of blood-brain barrier, we assessed the transendothelial migratory abilities of the different cTfh-cell subsets. Finally, we performed an RNA sequencing analysis of paired CSF cTfh cells and blood cTfh cells in 8 patients sampled at their first demyelinating event. RESULTS: The blood phenotype and frequency of cTfh cells were not significantly modified in patients with RRMS. In the CSF, we found an important infiltration of Tfh1 cells, with a high proportion of activated PD1+ cells. We demonstrated that the specific subset of Tfh1 cells presents increased migration abilities to cross an in vitro model of blood-brain barrier. Of interest, even at the first demyelinating event, cTfh cells in the CSF display specific characteristics with upregulation of EOMES gene and proinflammatory/cytotoxic transcriptomic signature able to efficiently distinguish cTfh cells from the CSF and blood. Finally, interactome analysis revealed potential strong cross talk between pathogenic B cells and CSF cTfh cells, pointing out the CSF as opportune supportive compartment and highlighting the very early implication of B-cell helper T cells in MS pathogenesis. DISCUSSION: Overall, CSF enrichment in activated Tfh1 as soon as disease diagnosis, associated with high expression of EOMES, and a predicted high propensity to interact with CSF B cells suggest that these cells probably contribute to disease onset and/or activity.

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Single-cell resolution of plasma cell fate programming in health and disease.

Long considered a homogeneous population dedicated to antibody secretion, plasma cell phenotypic and functional heterogeneity is increasingly recognized. Plasma cells were first segregated based on their maturation level, but the complexity of this subset might well be underestimated by this simple dichotomy. Indeed, in the last decade new functions have been attributed to plasma cells including but not limited to cytokine secretion. However, a proper characterization of plasma cell heterogeneity has remained elusive partly due to technical issues and cellular features that are specific to this cell type. Cell intrinsic and cell extrinsic signals could be at the origin of this heterogeneity. Recent advances in technologies such as single cell RNA-seq, ATAC-seq, or ChIP-seq on low cell numbers helped to elucidate the fate decision in other cell lineages and similar approaches could be implemented to evaluate the heterogeneous fate of activated B cells in health and disease. Here, we summarized published work shedding some lights on the stimuli and genetic program shaping B-cell terminal differentiation at the single cell level in mice and men. We also discuss the fate and heterogeneity of plasma cells during immune responses, vaccination, and in the frame of human plasma cell disorders.

Committed Human CD23-Negative Light-Zone Germinal Center B Cells Delineate Transcriptional Program Supporting Plasma Cell Differentiation.

B cell affinity maturation occurs in the germinal center (GC). Light-zone (LZ) GC B cells (BGC-cells) interact with follicular dendritic cells (FDCs) and compete for the limited, sequential help from T follicular helper cells needed to escape from apoptosis and complete their differentiation. The highest-affinity LZ BGC-cells enter the cell cycle and differentiate into PCs, following a dramatic epigenetic reorganization that induces transcriptome changes in general and the expression of the PRDM1 gene in particular. Human PC precursors are characterized by the loss of IL-4/STAT6 signaling and the absence of CD23 expression. Here, we studied the fate of human LZ BGC-cells as a function of their CD23 expression. We first showed that CD23 expression was restricted to the GC LZ, where it was primarily expressed by FDCs; less than 10% of tonsil LZ BGC-cells were positive. Sorted LZ BGC-cells left in culture and stimulated upregulated CD23 expression but were unable to differentiate into PCs - in contrast to cells that did not upregulate CD23 expression. An in-depth analysis (including single-cell gene expression) showed that stimulated CD23-negative LZ BGC-cells differentiated into plasmablasts and time course of gene expression changes delineates the transcriptional program that sustains PC differentiation. In particular, we identified a B cell proliferation signature supported by a transient MYC gene expression. Overall, the CD23 marker might be of value in answering questions about the differentiation of normal BGC-cells and allowed us to propose an instructive LZ BGC-cells maturation and fate model.

The diversity of the plasmablast signature across species and experimental conditions: A meta-analysis.

Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Locus suicide recombination actively occurs on the functionally rearranged IgH allele in B-cells from inflamed human lymphoid tissues.

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.

IL-2 imprints human naive B cell fate towards plasma cell through ERK/ELK1-mediated BACH2 repression.

Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps to reach the induction threshold. To further understand mechanisms by which T cell inputs regulate B cell fate decision, we investigate the minimal IL-2 stimulation for triggering human plasma cell differentiation in vitro. Here we show that the timed repression of BACH2 through IL-2-mediated ERK/ELK1 signalling pathway directs plasma cell lineage commitment. Enforced BACH2 repression in activated B cells unlocks the plasma cell transcriptional program and induces their differentiation into immunoglobulin M-secreting cells. RNA-seq and ChIP-seq results further identify BACH2 target genes involved in this process. An active regulatory region within the BACH2 super-enhancer, under ELK1 control and differentially regulated upon B-cell activation and cellular divisions, helps integrate IL-2 signal. Our study thus provides insights into the temporal regulation of BACH2 and its targets for controlling the differentiation of human naive B cells.

Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

Protocols for investigating microRNA functions in human neural progenitor cells.

Human embryonic stem cells and induced pluripotent stem cells offer great hope for studies of pathogenic mechanisms of disease and cell-based therapies. One powerful approach to manipulate the behaviors of human stem cells and their progenies is through microRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Each miRNA may target up to hundreds of mRNAs; some are specifically expressed in progenitor cells and affect multiple cellular processes. Here we present experimental protocols for investigating the endogenous functions of specific miRNAs in the proliferation, survival, and migration of human neural progenitor cells derived from embryonic stem cells. These methods may be applicable to protein factors and neural progenitor cells derived from patient-specific induced pluripotent stem cells.

IL-2 requirement for human plasma cell generation: coupling differentiation and proliferation by enhancing MAPK-ERK signaling.

Mature B cell differentiation involves a well-established transcription factor cascade. However, the temporal dynamics of cell signaling pathways regulating transcription factor network and coordinating cell proliferation and differentiation remain poorly defined. To gain insight into the molecular processes and extrinsic cues required for B cell differentiation, we set up a controlled primary culture system to differentiate human naive B cells into plasma cells (PCs). We identified T cell-produced IL-2 to be critically involved in ERK1/2-triggered PC differentiation. IL-2 drove activated B cell differentiation toward PC independently of its proliferation and survival functions. Indeed, IL-2 potentiated ERK activation and subsequent BACH2 and IRF8 downregulation, sustaining BLIMP1 expression, the master regulator for PC differentiation. Inhibition of the MAPK-ERK pathway, unlike STAT5 signaling, impaired IL-2-induced PC differentiation and rescued the expression profile of BACH2 and IRF8. These results identify IL-2 as a crucial early input in mature B cell fate commitment.

ESCRT-III subunits Snf7-1 and Snf7-2 differentially regulate transmembrane cargos in hESC-derived human neurons.

BACKGROUNDS: Endosomal sorting complex required for transport (ESCRT) is involved in several fundamental cellular processes and human diseases. Many mammalian ESCRT proteins have multiple isoforms but their precise functions remain largely unknown, especially in human neurons. RESULTS: In this study, we differentiated human embryonic stem cells (hESCs) into postmitotic neurons and characterized the functional properties of these neurons. Moreover, we found that among the three human paralogs of the yeast ESCRT-III subunit Snf7, hSnf7-1 and hSnf7-2 are most abundantly expressed in human neurons. Both hSnf7-1 and hSnf7-2 are required for the survival of human neurons, indicating a non-redundant essential function. Indeed, hSnf7-1 and hSnf7-2 are preferentially associated with CHMP2A and CHMP2B, respectively, and regulate the turnover of distinct transmembrane cargos such as neurotransmitter receptors in human neurons. CONCLUSION: These findings indicate that different mammalian paralogs of the yeast ESCRT-III subunit Snf7 have non-redundant functions in human neurons, suggesting that ESCRT-III with distinct subunit compositions may preferentially regulate different cargo proteins.

MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.

Human pluripotent stem cells offer promise for use in cell-based therapies for brain injury and diseases. However, their cellular behavior is poorly understood. Here we show that the expression of the brain-specific microRNA-9 (miR-9) is turned on in human neural progenitor cells (hNPCs) derived from human embryonic stem cells. Loss of miR-9 suppressed proliferation but promoted migration of hNPCs cultured in vitro. hNPCs without miR-9 activity also showed enhanced migration when transplanted into mouse embryonic brains or adult brains of a mouse model of stroke. These effects were not due to precocious differentiation of hNPCs. One of the key targets directly regulated by miR-9 encodes stathmin, which increases microtubule instability and whose expression in hNPCs correlates inversely with that of miR-9. Partial inhibition of stathmin activity suppressed the effects of miR-9 loss on proliferation and migration of human or embryonic rat neural progenitors. These results identify miR-9 as a novel regulator that coordinates the proliferation and migration of hNPCs.

Deletion of WNK1 first intron results in misregulation of both isoforms in renal and extrarenal tissues.

Large deletions in intron 1 of the with-no-lysine kinase type 1 (WNK1) gene cause familial hyperkalemic hypertension. Alternative promoters generate functionally different isoforms: long ubiquitous isoforms (L-WNK1) and a kidney-specific isoform (KS-WNK1) lacking kinase activity. It remains unclear whether the disease-causing mutations selectively modify the synthesis of 1 or both types of isoforms. Using a transgenic mouse model, we found that intron 1 deletion resulted in the overexpression of L- and KS-WNK1 in the distal convoluted tubule and ubiquitous ectopic KS-WNK1 expression. Phylogenetic and functional analysis of the minimal 22-kb intron 1 deletion identified 1 repressor and 1 insulator, potentially preventing interactions between the regulatory elements of L-WNK1 and KS-WNK1. These results provide the first insight into the molecular mechanisms of WNK1-induced familial hyperkalemic hypertension.

Cardiovascular expression of the mouse WNK1 gene during development and adulthood revealed by a BAC reporter assay.

Large deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1(-/-) mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation.