Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

1,8-cineole (eucalyptol): A versatile phytochemical with therapeutic applications across multiple diseases.

1,8-cineole (Eucalyptol), a naturally occurring compound derived from botanical sources such as eucalyptus, rosemary, and camphor laurel, has a long history of use in traditional medicine and exhibits an array of biological properties, including anti-inflammatory, antioxidant, antimicrobial, bronchodilatory, analgesic, and pro-apoptotic effects. Recent evidence has also indicated its potential role in managing conditions such as Alzheimer's disease, neuropathic pain, and cancer. This review spotlights the health advantages of 1,8-cineole, as demonstrated in clinical trials involving patients with respiratory disorders, including chronic obstructive pulmonary disease, asthma, bronchitis, and rhinosinusitis. In addition, we shed light on potential therapeutic applications of 1,8-cineole in various conditions, such as depression, epilepsy, peptic ulcer disease, diarrhea, cardiac-related heart diseases, and diabetes mellitus. A comprehensive understanding of 1,8-cineole's pharmacodynamics and safety aspects as well as developing effective formulations, might help to leverage its therapeutic value. This thorough review sets the stage for future research on diverse health benefits and potential uses of 1,8-cineole in tackling complex medical conditions.

The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1-/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing.

BACKGROUND: The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, its activity against earlier stages is limited. Current in vitro drug screening strategies depend on newly transformed schistosomula (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each intermediate larval stage, reducing the reliance on animal use (3Rs). METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and successfully cultured for up to four weeks with no loss in viability in a commercially available medium. Under these serum- and cell-free conditions, development halted at the lung-stage (LuS). However, the addition of human serum (HSe) propelled further development into liver stage (LiS) worms within eight weeks. Skin and lung stages, as well as LiS, were submitted to 96-well drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility to these compounds. CONCLUSION: With this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to LiS worms can be generated and maintained over prolonged periods of time. The phenotype of LiS worms, when exposed to reference drugs, was comparable to most previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in search for new anti-schistosomal drugs.

Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

Regulation of epithelial cell expressed C3 in the intestine - Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis. Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls. These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI.

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.

Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction.

Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.

Extracellular MRP8/14 is a regulator of ß2 integrin-dependent neutrophil slow rolling and adhesion.

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid ß2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.

T cell-dependence of Lassa fever pathogenesis.

Lassa virus (LASV), the causative agent of Lassa fever (LF), is endemic in West Africa, accounting for substantial morbidity and mortality. In spite of ongoing research efforts, LF pathogenesis and mechanisms of LASV immune control remain poorly understood. While normal laboratory mice are resistant to LASV, we report that mice expressing humanized instead of murine MHC class I (MHC-I) failed to control LASV infection and develop severe LF. Infection of MHC-I knockout mice confirmed a key role for MHC-I-restricted T cell responses in controlling LASV. Intriguingly we found that T cell depletion in LASV-infected HHD mice prevented disease, irrespective of high-level viremia. Widespread activation of monocyte/macrophage lineage cells, manifest through inducible NO synthase expression, and elevated IL-12p40 serum levels indicated a systemic inflammatory condition. The absence of extensive monocyte/macrophage activation in T cell-depleted mice suggested that T cell responses contribute to deleterious innate inflammatory reactions and LF pathogenesis. Our observations in mice indicate a dual role for T cells, not only protecting from LASV, but also enhancing LF pathogenesis. The possibility of T cell-driven enhancement and immunopathogenesis should be given consideration in future LF vaccine development.

Immune requirements of post-exposure immunization with modified vaccinia Ankara of lethally infected mice.

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer.

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity.

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Innate immune-induced depletion of bone marrow neutrophils aggravates systemic bacterial infections.

Neutrophils are the most abundant leukocytes in circulation and provide a primary innate immune defense function against bacterial pathogens before development of a specific immune response. These specialized phagocytes are short lived (12-24 hours) and continuously replenished from bone marrow. We found that if the host is overwhelmed by a high inoculum of Listeria monocytogenes, neutrophils are depleted despite high granulocyte-colony stimulating factor induction. In contrast to a low-dose innocuous L. monocytogenes infection, high-dose Listeria challenge blocks neutrophil recruitment to infectious abscesses and bacterial proliferation is not controlled, resulting in lethal outcomes. Administering synthetic TLR2-ligand or heat-killed bacteria during the innocuous L. monocytogenes infection reproduced these effects, once again leading to overwhelming bacterial propagation. The same stimuli also severely aggravated Salmonella typhimurium, Staphylococcus aureus, and Streptococcus pyogenes systemic infection. These data implicate systemic innate immune stimulation as a mechanism of bone marrow neutrophil exhaustion which negatively influences the outcome of bacterial infections.

Hematopoietic cell-derived interferon controls viral replication and virus-induced disease.

Type I interferon (IFN-I) strongly inhibits viral replication and is a crucial factor in controlling virus infections and diseases. Cellular activation through pattern recognition receptors induces interferon production in a wide variety of hematopoietic and nonhematopoietic cell types, including dendritic cells, fibroblasts, hepatocytes, and cells of neuronal origin. The relative contribution of hematopoietic and nonhematopoietic cells to the overall interferon response is an important issue which has not been fully addressed. Using irf7(-/-) and wild-type bone marrow chimeras we analyzed the contribution of IFN-I from bone marrow-derived sources in the control of viral infections and immunopathology in mice. We found that during systemic cytopathic virus infection, hematopoietic cells were essential for production of IFN-I, inhibition of viral spread to peripheral organs, and limiting cell damage. In a model of autoimmune diabetes induced by noncytopathic virus infection, hematopoietic cell-derived IFN-I was essential for CD8(+) T cell-dependent cytotoxicity in pancreatic beta-islet cells and induction of diabetes. These data suggest that during systemic viral infection primarily hematopoietic cell-derived IFN-I controls viral replication and viral-induced disease.

Circulating C3 is necessary and sufficient for induction of autoantibody-mediated arthritis in a mouse model.

OBJECTIVE: For the inflammation characteristic of rheumatoid arthritis, the relative contribution of mediators produced locally in the synovium versus those circulating systemically is unknown. Complement factor C3 is made in rheumatoid synovium and has been proposed to be a crucial driver of inflammation. The aim of this study was to test, in a mouse model of rheumatoid arthritis, whether C3 synthesized within the synovium is important in promoting inflammation. METHODS: Radiation bone marrow chimeras between normal and C3(-/-) mice were constructed in order to generate animals that expressed or lacked expression of C3 only in hematopoietic cells. Parabiotic mice were made by surgically linking C3(-/-) mice to irradiated wild-type mice to obtain animals having C3 only in the circulation. Arthritis was induced by injection of serum from arthritic K/BxN mice. RESULTS: In bone marrow chimeras, synthesis of C3 by radioresistant cells was necessary and sufficient to confer susceptibility to serum-transferred arthritis. Parabionts having C3 only in the circulation remained sensitive to arthritis induction, and the cartilage of these arthritic mice contained deposits of C3. CONCLUSION: In a mouse model in which the alternative pathway of complement activation is critical to the induction of arthritis by autoantibodies, circulating C3 was necessary and sufficient for arthritis induction.

Follicular exclusion of autoreactive B cells requires FcgammaRIIb.

In non-autoimmune mice, the 3H9 transgenic Ig heavy chain can pair with endogenous Iglambda1 light chains to generate B cells with specificity for DNA. These autoreactive cells are actively regulated in vivo, as indicated by the exclusion of lambda1 cells from the splenic B cell follicle and the absence of auto-antibody production. To study the role of Fcgamma receptor IIb (FcgammaRIIb) in peripheral B cell tolerance, FcgammaRIIb(-/-) mice were crossed with C57BL/6 mice bearing a site-directed knock-in of the 3H9 transgene. 3H9FcgammaRIIb(-/-) mice become autoreactive, lose the follicular exclusion of anti-DNA B cells and instead have lambda1 B cells located within splenic germinal centers. They have increased frequencies of splenic auto-antibody-producing cells and elevated titers of IgG anti-DNA auto-antibody. The data implicate an FcgammaRIIb-dependent checkpoint that can exclude autoreactive B cells from splenic follicles. By restricting their participation in germinal center reactions, this putative checkpoint helps attenuate the production of potentially pathogenic auto-antibodies. The data further suggest that this FcgammaRIIb-dependent regulation is B cell autonomous.

Optimal long-term humoral responses to replication-defective herpes simplex virus require CD21/CD35 complement receptor expression on stromal cells.

Replication-defective herpes simplex virus (HSV) strains elicit durable immune responses and protect against virulent HSV challenge in mice, despite being unable to establish latent infection in neuronal cells. Mechanisms for generating long-lived immunity in the absence of viral persistence remain uncertain. In animals immunized with replication-defective HSV, durable serum immunoglobulin G (IgG) responses were elicited. Surprisingly, Western blot analyses revealed that the specificities of antiviral IgG changed over time, and antibody reactivity to some viral proteins was detected only very late. Thus, some of the durable IgG activity appeared to be contributed by either new or significantly enhanced antibody responses at late times. Following immunization, radiation bone marrow-chimeric mice lacking complement receptors CD21 and CD35 on stromal cells elicited only short-lived serum IgG and failed to mount recall responses to subsequent HSV exposure. Our results suggest that complement-mediated retention of viral antigens by stromal cells, such as follicular dendritic cells, is critical for optimal maintenance of antibody responses and B-cell memory following vaccination with replication-defective HSV.

Myeloid C3 determines induction of humoral responses to peripheral herpes simplex virus infection.

The complement system, in addition to its role in innate immunity, is an important regulator of the B cell response. Complement exists predominantly in the circulation and although the primary source is hepatic, multiple additional cellular sources have been described that can contribute substantially to the complement pool. To date, however, complement produced by these secondary sources has been deemed redundant to that secreted by the liver. In contrast, using a bone marrow chimeric model, we observed that C3 synthesis by myeloid cells, a relatively minor source of complement, provided a critical function during the induction of humoral responses to peripheral HSV infection. Anti-viral Ab, as generated in an efficient humoral response, has been associated with protection from severe consequences of HSV dissemination. This report offers insight into the generation of the adaptive immune response in the periphery and describes a unique role for a nonhepatic complement source.

Macrophage-derived complement component C4 can restore humoral immunity in C4-deficient mice.

Mice with a disrupted C4 locus (C4(-/-)) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl(5) conjugated to keyhole limpet hemocyanin (NP(5)- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP(5)-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b(+), CD11c(-)) as a cellular source for C4 synthesis within the spleen.