Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Follicular dendritic cells emerge from ubiquitous perivascular precursors.

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor ß (PDGFRß). PDGFRß-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRß(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRß(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRß(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin ß receptor (LTßR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIß(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRß(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection.

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation.

Platelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI.

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbß3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

Impaired function and delayed regeneration of dendritic cells in COVID-19.

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Predicting single-cell gene expression profiles of imaging flow cytometry data with machine learning.

High-content imaging and single-cell genomics are two of the most prominent high-throughput technologies for studying cellular properties and functions at scale. Recent studies have demonstrated that information in large imaging datasets can be used to estimate gene mutations and to predict the cell-cycle state and the cellular decision making directly from cellular morphology. Thus, high-throughput imaging methodologies, such as imaging flow cytometry can potentially aim beyond simple sorting of cell-populations. We introduce IFC-seq, a machine learning methodology for predicting the expression profile of every cell in an imaging flow cytometry experiment. Since it is to-date unfeasible to observe single-cell gene expression and morphology in flow, we integrate uncoupled imaging data with an independent transcriptomics dataset by leveraging common surface markers. We demonstrate that IFC-seq successfully models gene expression of a moderate number of key gene-markers for two independent imaging flow cytometry datasets: (i) human blood mononuclear cells and (ii) mouse myeloid progenitor cells. In the case of mouse myeloid progenitor cells IFC-seq can predict gene expression directly from brightfield images in a label-free manner, using a convolutional neural network. The proposed method promises to add gene expression information to existing and new imaging flow cytometry datasets, at no additional cost.

The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles.

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Follicular dendritic cells: origin, phenotype, and function in health and disease.

Follicular dendritic cells (FDCs) were originally identified by their specific morphology and by their ability to trap immune-complexed antigen in B cell follicles. By virtue of the latter as well as the provision of chemokines, adhesion molecules, and trophic factors, FDCs participate in the shaping of B cell responses. Importantly, FDCs also supply tingible body macrophages (TBMs) with the eat-me-signaling molecule milk fat globule-EGF factor 8 (Mfge8), thereby enabling the disposal of apoptotic B cells. Recent studies have provided fundamental insights into the multiple functions of FDCs in both physiological and pathophysiological contexts and into their origin. Here we review these findings, and discuss current concepts related to FDC histogenesis both in lymphoid organs and in inflammatory lymphoneogenesis.

Lymphotoxin-dependent prion replication in inflammatory stromal cells of granulomas.

Prior to invading the nervous system, prions frequently colonize lymphoid organs and sites of inflammatory lymphoneogenesis, where they colocalize with Mfge8+ follicular dendritic cells (FDCs). Here, we report that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LTbetaR), even though they lacked FDCs and did not display lymphoneogenesis. After intraperitoneal prion inoculation, granulomas of Prnp(+/+) mice, but not Prnp(-/-) granulomas or unaffected Prnp(+/+) skin, accumulated prion infectivity and disease-associated prion protein. Bone-marrow transfers between Prnp(+/+) and Prnp(-/-) mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and LTbetaR signaling. Granulomatous PrPC was mainly expressed by stromal LTbetaR+ mesenchymal cells that were absent from unaffected subcutis. Hence, granulomas can act as clinically silent reservoirs of prion infectivity. Furthermore, lymphotoxin-dependent prion replication can occur in inflammatory stromal cells that are distinct from FDCs.

Microbial influences on epithelial integrity and immune function as a basis for inflammatory diseases.

Certain autoimmune diseases as well as asthma have increased in recent decades, particularly in developed countries. The hygiene hypothesis has been the prevailing model to account for this increase; however, epidemiology studies also support the contribution of diet and obesity to inflammatory diseases. Diet affects the composition of the gut microbiota, and recent studies have identified various molecules and mechanisms that connect diet, the gut microbiota, and immune responses. Herein, we discuss the effects of microbial metabolites, such as short chain fatty acids, on epithelial integrity as well as immune cell function. We propose that dysbiosis contributes to compromised epithelial integrity and disrupted immune tolerance. In addition, dietary molecules affect the function of immune cells directly, particularly through lipid G-protein coupled receptors such as GPR43.

Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.

The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in 'healthy' microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA-GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43(-/-)) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43(-/-) immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

Commensal flora and the regulation of inflammatory and autoimmune responses.

The gut microbiota has recently been recognized for its role in immune regulation, and changes in gut microbiota may be the basis for an increased incidence of autoimmune diseases and asthma in developed countries. Beneficial microbes produce factors that are distributed systemically, and therefore can influence peripheral inflammatory responses. Such symbiosis factors are important for the control and resolution of inflammation and autoimmune diseases. Here we discuss immune regulation by recently identified symbiosis factors and how certain environmental factors favor their production and influence the composition of the gut microflora.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

The small molecule inhibitor BX-795 uncouples IL-2 production from inhibition of Th2 inflammation and induces CD4+ T cells resembling iTreg.

Background: Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development. Objective: To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds. Materials and methods: We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds. Results: We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils. Conclusions: BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases.