Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity.

BACKGROUND: Recent evidence has shown dopamine as a major regulator of inflammation. Accordingly, dopaminergic regulation of immune cells plays an important role in the physiopathology of inflammatory disorders. Multiple sclerosis (MS) is an inflammatory disease involving a CD4+ T-cell-driven autoimmune response to central nervous system (CNS) derived antigens. Evidence from animal models has suggested that B cells play a fundamental role as antigen-presenting cells (APC) re-stimulating CD4+ T cells in the CNS as well as regulating T-cell response by mean of inflammatory or anti-inflammatory cytokines. Here, we addressed the role of the dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in B cells in animal models of MS. METHODS: Mice harbouring Drd3-deficient or Drd3-sufficient B cells were generated by bone marrow transplantation into recipient mice devoid of B cells. In these mice, we compared the development of experimental autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein (MOG)-derived peptide (pMOG), a model that leads to CNS-autoimmunity irrespective of the APC-function of B cells, or by immunization with full-length human MOG protein (huMOG), a model in which antigen-specific activated B cells display a fundamental APC-function in the CNS. APC-function was assessed in vitro by pulsing B cells with huMOG-coated beads and then co-culturing with MOG-specific T cells. RESULTS: Our data show that the selective Drd3 deficiency in B cells abolishes the disease development in the huMOG-induced EAE model. Mechanistic analysis indicates that although DRD3-signalling did not affect the APC-function of B cells, DRD3 favours the CNS-tropism in a subset of pro-inflammatory B cells in the huMOG-induced EAE model, an effect that was associated with higher CXCR3 expression. Conversely, the results show that the selective Drd3 deficiency in B cells exacerbates the disease severity in the pMOG-induced EAE model. Further analysis shows that DRD3-stimulation increased the expression of the CNS-homing molecule CD49d in a B-cell subset with anti-inflammatory features, thus attenuating EAE manifestation in the pMOG-induced EAE model. CONCLUSIONS: Our findings demonstrate that DRD3 in B cells exerts a dual role in CNS-autoimmunity, favouring CNS-tropism of pro-inflammatory B cells with APC-function and promoting CNS-homing of B cells with anti-inflammatory features. Thus, these results show DRD3-signalling in B cells as a critical regulator of CNS-autoimmunity.

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.

Endolysosomal vesicles at the center of B cell activation.

The endolysosomal system specializes in degrading cellular components and is crucial to maintaining homeostasis and adapting rapidly to metabolic and environmental cues. Cells of the immune system exploit this network to process antigens or promote cell death by secreting lysosome-related vesicles. In B lymphocytes, lysosomes are harnessed to facilitate the extraction of antigens and to promote their processing into peptides for presentation to T cells, critical steps to mount protective high-affinity antibody responses. Intriguingly, lysosomal vesicles are now considered important signaling units within cells and also display secretory functions by releasing their content to the extracellular space. In this review, we focus on how B cells use pathways involved in the intracellular trafficking, secretion, and function of endolysosomes to promote adaptive immune responses. A basic understanding of such mechanisms poses an interesting frontier for the development of therapeutic strategies in the context of cancer and autoimmune diseases.

Medium-throughput image-based phenotypic siRNA screen to unveil the molecular basis of B cell polarization.

Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.

Clinical and pulmonary function analysis in long-COVID revealed that long-term pulmonary dysfunction is associated with vascular inflammation pathways and metabolic syndrome.

Introduction: Long-term pulmonary dysfunction (L-TPD) is one of the most critical manifestations of long-COVID. This lung affection has been associated with disease severity during the acute phase and the presence of previous comorbidities, however, the clinical manifestations, the concomitant consequences and the molecular pathways supporting this clinical condition remain unknown. The aim of this study was to identify and characterize L-TPD in patients with long-COVID and elucidate the main pathways and long-term consequences attributed to this condition by analyzing clinical parameters and functional tests supported by machine learning and serum proteome profiling. Methods: Patients with L-TPD were classified according to the results of their computer-tomography (CT) scan and diffusing capacity of the lungs for carbon monoxide adjusted for hemoglobin (DLCOc) tests at 4 and 12-months post-infection. Results: Regarding the acute phase, our data showed that L-TPD was favored in elderly patients with hypertension or insulin resistance, supported by pathways associated with vascular inflammation and chemotaxis of phagocytes, according to computer proteomics. Then, at 4-months post-infection, clinical and functional tests revealed that L-TPD patients exhibited a restrictive lung condition, impaired aerobic capacity and reduced muscular strength. At this time point, high circulating levels of platelets and CXCL9, and an inhibited FCgamma-receptor-mediated-phagocytosis due to reduced FcγRIII (CD16) expression in CD14+ monocytes was observed in patients with L-TPD. Finally, 1-year post infection, patients with L-TPD worsened metabolic syndrome and augmented body mass index in comparison with other patient groups. Discussion: Overall, our data demonstrated that CT scan and DLCOc identified patients with L-TPD after COVID-19. This condition was associated with vascular inflammation and impair phagocytosis of virus-antibody immune complexes by reduced FcγRIII expression. In addition, we conclude that COVID-19 survivors required a personalized follow-up and adequate intervention to reduce long-term sequelae and the appearance of further metabolic diseases.

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation.

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.

Autophagy Induced by Toll-like Receptor Ligands Regulates Antigen Extraction and Presentation by B Cells.

The engagement of B cells with surface-tethered antigens triggers the formation of an immune synapse (IS), where the local secretion of lysosomes can facilitate antigen uptake. Lysosomes intersect with other intracellular processes, such as Toll-like Receptor (TLR) signaling and autophagy coordinating immune responses. However, the crosstalk between these processes and antigen presentation remains unclear. Here, we show that TLR stimulation induces autophagy in B cells and decreases their capacity to extract and present immobilized antigens. We reveal that TLR stimulation restricts lysosome repositioning to the IS by triggering autophagy-dependent degradation of GEF-H1, a Rho GTP exchange factor required for stable lysosome recruitment at the synaptic membrane. GEF-H1 degradation is not observed in B cells that lack αV integrins and are deficient in TLR-induced autophagy. Accordingly, these cells show efficient antigen extraction in the presence of TLR stimulation, confirming the role of TLR-induced autophagy in limiting antigen extraction. Overall, our results suggest that resources associated with autophagy regulate TLR and BCR-dependent functions, which can finetune antigen uptake by B cells. This work helps to understand the mechanisms by which B cells are activated by surface-tethered antigens in contexts of subjacent inflammation before antigen recognition, such as sepsis.

Dynamics of membrane trafficking downstream of B and T cell receptor engagement: impact on immune synapses.

The onset of an adaptive immune response requires the activation of T and B lymphocytes by antigen-presenting cells, through a specialized form of intercellular communication, known as the immunological synapse (IS). In B lymphocytes the IS promotes efficient recognition and acquisition of membrane-bound Ags, while in T cells, it modulates the T cell response upon exposure to peptide-major histocompatibility complexes. In this review, we highlight the similarities that determine B and T cell activation, focusing on immune receptor downstream signaling events that lead to synapse formation. We stress the notion that polarization of T and B lymphocytes characterized by global changes in cytoskeleton and membrane trafficking modulates synapse structure and function, thus determining lymphocyte effector functions and fate.

Alterations in Lysosome Homeostasis in Lipid-Related Disorders: Impact on Metabolic Tissues and Immune Cells.

Lipid-related disorders, which primarily affect metabolic tissues, including adipose tissue and the liver are associated with alterations in lysosome homeostasis. Obesity is one of the more prevalent diseases, which results in energy imbalance within metabolic tissues and lysosome dysfunction. Less frequent diseases include Niemann-Pick type C (NPC) and Gaucher diseases, both of which are known as Lysosomal Storage Diseases (LSDs), where lysosomal dysfunction within metabolic tissues remains to be fully characterized. Adipocytes and hepatocytes share common pathways involved in the lysosome-autophagic axis, which are regulated by the function of cathepsins and CD36, an immuno-metabolic receptor and display alterations in lipid diseases, and thereby impacting metabolic functions. In addition to intrinsic defects observed in metabolic tissues, cells of the immune system, such as B cells can infiltrate adipose and liver tissues, during metabolic imbalance favoring inflammation. Moreover, B cells rely on lysosomes to promote the processing and presentation of extracellular antigens and thus could also present lysosome dysfunction, consequently affecting such functions. On the other hand, growing evidence suggests that cells accumulating lipids display defective inter-organelle membrane contact sites (MCSs) established by lysosomes and other compartments, which contribute to metabolic dysfunctions at the cellular level. Overall, in this review we will discuss recent findings addressing common mechanisms that are involved in lysosome dysregulation in adipocytes and hepatocytes during obesity, NPC, and Gaucher diseases. We will discuss whether these mechanisms may modulate the function of B cells and how inter-organelle contacts, emerging as relevant cellular mechanisms in the control of lipid homeostasis, have an impact on these diseases.

Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse.

The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findings support a model where B the asymmetric distribution of the proteasome, mediated by Ecm29, coordinates actin dynamics at the centrosome and the IS, promoting lysosome recruitment and cell spreading.

B Cells Adapt Their Nuclear Morphology to Organize the Immune Synapse and Facilitate Antigen Extraction.

Upon interaction with immobilized antigens, B cells form an immune synapse where actin remodeling and re-positioning of the microtubule-organizing center (MTOC) together with lysosomes can facilitate antigen extraction. B cells have restricted cytoplasmic space, mainly occupied by a large nucleus, yet the role of nuclear morphology in the formation of the immune synapse has not been addressed. Here we show that upon activation, B cells re-orientate and adapt the size of their nuclear groove facing the immune synapse, where the MTOC sits, and lysosomes accumulate. Silencing the nuclear envelope proteins Nesprin-1 and Sun-1 impairs nuclear reorientation towards the synapse and leads to defects in actin organization. Consequently, B cells are unable to internalize the BCR after antigen activation. Nesprin-1 and Sun-1-silenced B cells also fail to accumulate the tethering factor Exo70 at the center of the synaptic membrane and display defective lysosome positioning, impairing efficient antigen extraction at the immune synapse. Thus, changes in nuclear morphology and positioning emerge as critical regulatory steps to coordinate B cell activation.

Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes.

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit micro1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in approximately 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor.

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.

Studying MHC Class II Presentation of Immobilized Antigen by B Lymphocytes.

The ability of B lymphocytes to capture external antigens (Ag) and present them as peptide fragments, loaded on major histocompatibility complex (MHC) class II molecules, to CD4+ T cells is a crucial part of the adaptive immune response. This allows for T-B cooperation, a cellular communication that is required for B cells to develop into germinal centers (GC) and form mature high affinity antibody producing cells and to further develop B cell memory. MHC class II antigen presentation by B lymphocytes is a multistep process involving (1) Recognition and capture of external Ag by B lymphocytes through their B cell receptor (BCR), (2) Ag processing, which comprises the degradation of Ag in internal compartments within the B cell and loading of the corresponding peptide fragments on MHC class II molecules, and (3) Presentation of MHCII-peptide complexes to CD4+ T cells. Here, we describe how to study the biochemical and morphological changes that occur in B lymphocytes at these three major levels.

Studying Organelle Dynamics in B Cells During Immune Synapse Formation.

Recognition of surface-tethered antigens by the B cell receptor (BCR) triggers the formation of an immune synapse (IS), where both signaling and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Proteasome Dependent Actin Remodeling Facilitates Antigen Extraction at the Immune Synapse of B Cells.

Engagement of the B cell receptor (BCR) with surface-tethered antigens leads to the formation of an immune synapse (IS), where cell signaling and antigen uptake are tightly coordinated. Centrosome re-orientation to the immune synapse has emerged as a critical regulatory step to guide the local recruitment and secretion of lysosomes, which can facilitate the extraction of immobilized antigens. This process is coupled to actin remodeling at the centrosome and at the immune synapse, which is crucial to promote cell polarity. How B cells balance both pools of actin cytoskeleton to achieve a polarized phenotype during the formation of an immune synapse is not fully understood. Here, we reveal that B cells rely on proteasome activity to achieve this task. The proteasome is a multi-catalytic protease that degrades cytosolic and nuclear proteins and its dysfunction is associated with diseases, such as cancer and autoimmunity. Our results show that resting B cells contain an active proteasome pool at the centrosome, which is required for efficient actin clearance at this level. As a result of proteasome inhibition, activated B cells do not deplete actin at the centrosome and are unable to separate the centrosome from the nucleus and thus display impaired polarity. Consequently, lysosome recruitment to the immune synapse, antigen extraction and presentation are severely compromised in B cells with diminished proteasome activity. Additionally, we found that proteasome inhibition leads to impaired actin remodeling at the immune synapse, where B cells display defective spreading responses and distribution of key signaling molecules at the synaptic membrane. Overall, our results reveal a new role for the proteasome in regulating the immune synapse of B cells, where the intracellular compartmentalization of proteasome activity controls cytoskeleton remodeling between the centrosome and synapse, with functional repercussions in antigen extraction and presentation.

Lysosome motility and distribution: Relevance in health and disease.

Lysosomes are dynamic organelles, which can fuse with a variety of targets and undergo constant regeneration. They can move along microtubules in a retrograde and anterograde fashion by using motor proteins, kinesin and dynein, being main players in extracellular secretion, intracellular components degradation and recycling. Moreover, lysosomes interact with other intracellular organelles to regulate their turnover, such as ER, mitochondria and peroxisomes. The correct localization of lysosomes is relevant in several physiological processes, including appropriate antigen presentation, neurotransmission and receptors modulation in neuronal synapsis, whereas hepatic lysosomes and autophagy are master regulators of nutrient homeostasis. Alterations in lysosome function due to mutation of genes encoding lysosomal proteins, soluble hydrolases as well as membrane proteins, lead to lysosomal storage diseases (LSDs). Lysosomes containing undegraded substrates are finally stacked and therefore miss positioned inside the cell, leading to lysosomal dysfunction, which impacts a wide range of cellular functions.

KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover.

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.

The exocyst controls lysosome secretion and antigen extraction at the immune synapse of B cells.

B lymphocytes capture antigens from the surface of presenting cells by forming an immune synapse. Local secretion of lysosomes, which are guided to the synaptic membrane by centrosome repositioning, can facilitate the extraction of immobilized antigens. However, the molecular basis underlying their delivery to precise domains of the plasma membrane remains elusive. Here we show that microtubule stabilization, triggered by engagement of the B cell receptor, acts as a cue to release centrosome-associated Exo70, which is redistributed to the immune synapse. This process is coupled to the recruitment and activation of GEF-H1, which is required for assembly of the exocyst complex, used to promote tethering and fusion of lysosomes at the immune synapse. B cells silenced for GEF-H1 or Exo70 display defective lysosome secretion, which results in impaired antigen extraction and presentation. Thus, centrosome repositioning coupled to changes in microtubule stability orchestrates the spatial-temporal distribution of the exocyst complex to promote polarized lysosome secretion at the immune synapse.

Tuning B cell responses to antigens by cell polarity and membrane trafficking.

The capacity of B lymphocytes to produce specific antibodies, particularly broadly neutralizing antibodies that provide immunity to viral pathogens has positioned them as valuable therapeutic targets for immunomodulation. To become competent as antibody secreting cells, B cells undergo a series of activation steps, which are triggered by the recognition of antigens frequently displayed on the surface of other presenting cells. Such antigens elicit the formation of an immune synapse (IS), where local cytoskeleton rearrangements coupled to mechanical forces and membrane trafficking orchestrate the extraction and processing of antigens in B cells. In this review, we discuss the molecular mechanisms that regulate polarized membrane trafficking and mechanical properties of the immune synapse, as well as the potential extracellular cues from the environment, which may impact the ability of B cells to sense and acquire antigens at the immune synapse. An integrated view of the diverse cellular mechanisms that shape the immune synapse will provide a better understanding on how B cells are efficiently activated.