Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.

Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota.

Mononuclear phagocytes are an important component of an innate immune system perceived as a system ready to react upon encounter of pathogens. Here, we show that in response to microbial stimulation, mononuclear phagocytes residing in nonmucosal lymphoid organs of germ-free mice failed to induce expression of a set of inflammatory response genes, including those encoding the various type I interferons (IFN-I). Consequently, NK cell priming and antiviral immunity were severely compromised. Whereas pattern recognition receptor signaling and nuclear translocation of the transcription factors NF-κB and IRF3 were normal in mononuclear phagocytes of germ-free mice, binding to their respective cytokine promoters was impaired, which correlated with the absence of activating histone marks. Our data reveal a previously unrecognized role for postnatally colonizing microbiota in the introduction of chromatin level changes in the mononuclear phagocyte system, thereby poising expression of central inflammatory genes to initiate a powerful systemic immune response during viral infection.

TLR2 Stimulation Increases Cellular Metabolism in CD8+ T Cells and Thereby Enhances CD8+ T Cell Activation, Function, and Antiviral Activity.

TLR2 serves as a costimulatory molecule on activated T cells. However, it is unknown how the functionality and antiviral activity of CD8+ T cells are modulated by direct TLR2 signaling. In this study, we looked at the TLR2-mediated enhancement of TCR-driven CD8+ T cell activation in vitro and in woodchuck hepatitis virus transgenic mice. In vitro stimulation of CD8+ T cells purified from C57BL/6 mice showed that TLR2 agonist Pam3CSK4 directly enhanced the TCR-dependent CD8+ T cell activation. Transcriptome analysis revealed that TLR2 signaling increased expression of bioenergy metabolism-related genes in CD8+ T cells, such as IRF4, leading to improved glycolysis and glutaminolysis. This was associated with the upregulation of genes related to immune regulation and functions such as T-bet and IFN-γ. Glycolysis and glutaminolysis were in turn essential for the TLR2-mediated enhancement of T cell activation. Administration of TLR2 agonist Pam3CSK4 promoted the expansion and functionality of vaccine-primed, Ag-specific CD8+ T cells in both wild type and transgenic mice and improved viral suppression. Thus, TLR2 could promote CD8+ T cell immunity through regulating the energy metabolism.

Mycobacteria exploit nitric oxide-induced transformation of macrophages into permissive giant cells.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion.

Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to ß-hemolytic streptococci.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

BACKGROUND: Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. OBJECTIVE: We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. METHODS: L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4+ T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. RESULTS: L lactis G121-treated murine BMDCs and human moDCs released TH1-polarizing cytokines and induced TH1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of TH1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13-/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The TH1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. CONCLUSION: Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8.

Toll-Like Receptor 2 and Mincle Cooperatively Sense Corynebacterial Cell Wall Glycolipids.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

Staphylococcus aureus-derived lipoteichoic acid induces temporary T-cell paralysis independent of Toll-like receptor 2.

BACKGROUND: The interplay between microbes and surface organs, such as the skin, shapes a complex immune system with several checks and balances. The first-line defense is mediated by innate immune pathways leading to inflammation. In the second phase specific T cells invade the infected organ, amplifying inflammation and defense. Consecutively, termination of inflammation is crucial to avoid chronic inflammation triggered by microbes, such as in patients with atopic dermatitis. OBJECTIVE: We aimed to elucidate how the Staphylococcus aureus-derived cell-wall component lipoteichoic acid (LTA) governs the second phase of immune responses when high concentrations of LTA access T cells directly through disrupted skin. METHODS: We analyzed the direct exposure of T cells to LTA in vitro. For in vivo analyses, we used fluorescein isothiocyanate contact hypersensitivity and ovalbumin-induced dermatitis as models for TH2-mediated cutaneous inflammation. RESULTS: We observed that LTA potently suppressed T-lymphocyte activation in a Toll-like receptor 2-independent manner. LTA-exposed T cells did not proliferate and did not produce cytokines. Importantly, these T cells remained completely viable and were responsive to consecutive activation signals on subsequent removal of LTA. Thus LTA exposure resulted in temporary functional T-cell paralysis. In vivo experiments revealed that T-cell cytokine production and cutaneous recall responses were significantly suppressed by LTA. CONCLUSION: We identified a new mechanism through which bacterial compounds directly but temporarily modulate adaptive immune responses.

Expression of type I interferon by splenic macrophages suppresses adaptive immunity during sepsis.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.

RAIDD Mediates TLR3 and IRF7 Driven Type I Interferon Production.

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Chemokine (C-C Motif) receptor 1 is required for efficient recruitment of neutrophils during respiratory infection with modified vaccinia virus Ankara.

UNLABELLED: Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1(-/-) mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE: Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

Bacterial RNA mediates activation of caspase-1 and IL-1ß release independently of TLRs 3, 7, 9 and TRIF but is dependent on UNC93B.

Recognition of foreign nucleic acids is important for the induction of an innate immune response against invading pathogens. Although the pathways involved in sensing bacterial DNA and viral RNA are now well established, only limited knowledge is available on mechanisms underlying recognition of bacterial RNA. It has been reported that intracellular delivery of Escherichia coli RNA activates the Nlrp3 inflammasome, but whether this is a general property of bacterial RNA remains unclear as are the pathways involved in pro-IL-1ß induction and caspase-1 activation by bacterial RNA. In this study, we report that bacterial RNA from both Gram-positive and Gram-negative bacteria induces activation of caspase-1 and secretion of IL-1ß by murine dendritic cells and bone-marrow derived macrophages. Stimulation was independent of the presence of 5'-triphosphate termini and occurred with whole RNA preparations from bacteria but not from eukaryotes. Induction of pro-IL-1ß as well as the priming for caspase-1 activation by bacterial RNA was dependent on UNC93B, an endoplasmic reticulum protein essential for delivery of TLRs to the endosome, whereas the established nucleic acid sensing endosomal TLRs 3, 7, and 9 were dispensable. Additionally, caspase-1 activation and IL-1ß production by transfected bacterial RNA were absent in MyD88-deficient cells but independent of TRIF. Thus, our data indicate the presence of a yet unidentified intracellular nucleic acid receptor involved in bacterial RNA-induced inflammasome activation and release of IL-1ß.

Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection.

Retroviral infections e.g. HIV still represent a unique burden in the field of vaccine research. A common challenge in vaccine design is to find formulations that create appropriate immune responses to protect against and/or control the given pathogen. Nanoparticles have been considered to be ideal vaccination vehicles that mimic invading pathogens. In this study, we present biodegradable calcium phosphate (CaP) nanoparticles, functionalized with CpG and retroviral T cell epitopes of Friend virus (FV) as excellent vaccine delivery system. CaP nanoparticles strongly increased antigen delivery to antigen-presenting cells to elicit a highly efficient T cell-mediated immune response against retroviral FV infection. Moreover, single-shot immunization of chronically FV-infected mice with functionalized CaP nanoparticles efficiently reactivated effector T cells which led to a significant decrease in viral loads. Thus, our findings clearly indicate that a nanoparticle-based peptide immunization is a promising approach to improve antiretroviral vaccination. FROM THE CLINICAL EDITOR: In this study, biodegradable calcium phosphate nanoparticles were used as a vaccine delivery system after functionalization with CpG and Friend virus-derived T-cell epitopes. This vaccination strategy resulted in increased T-cell mediated immune response even in chronically infected mice, providing a promising approach to the development of clinically useful antiretroviral vaccination strategies.

Combining antibiotic with anti-TLR2/TLR13 therapy prevents brain pathology in pneumococcal meningitis.

Despite effective antibiotic therapy, brain-destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR-recruited myeloid differentiation primary response adaptor 88 (MyD88), as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and single-stranded RNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2-/- Tlr13-/- mice. Their phenotypic resemblance indicated TLR2 and -13 as master sensors of S. pneumoniae in the cerebrospinal fluid. A neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) - the latter applied here as an inhibitor of murine TLR13 and its human ortholog TLR8 - abrogated activation of murine and human primary immune cells exposed to antibiotic-treated S. pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/TLR13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared with treatment with ceftriaxone alone or in combination with dexamethasone. Our study indicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins.

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.

The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release.

Streptococcus pneumoniae meningitis causes brain damage through inflammation-related pathways whose identity and mechanisms of action are yet unclear. We previously identified caspase-1, which activates precursor IL-1 type cytokines, as a central mediator of inflammation in pneumococcal meningitis. In this study, we demonstrate that lack of the inflammasome components ASC or NLRP3 that are centrally involved in caspase-1 activation decreases scores of clinical and histological disease severity as well as brain inflammation in murine pneumococcal meningitis. Using specific inhibitors (anakinra and rIL-18-binding protein), we further show that ASC- and NLRP3-dependent pathologic alterations are solely related to secretion of both IL-1ß and IL-18. Moreover, using differentiated human THP-1 cells, we demonstrate that the pneumococcal pore-forming toxin pneumolysin is a key inducer of IL-1ß expression and inflammasome activation upon pneumococcal challenge. The latter depends on the release of ATP, lysosomal destabilization (but not disruption), and cathepsin B activation. The in vivo importance of this pathway is supported by our observation that the lack of pneumolysin and cathepsin B inhibition is associated with a better clinical course and less brain inflammation in murine pneumococcal meningitis. Collectively, our study indicates a central role of the NLRP3 inflammasome in the pathology of pneumococcal meningitis. Thus, interference with inflammasome activation might be a promising target for adjunctive therapy of this disease.

CCL17 controls mast cells for the defense against filarial larval entry.

Filarial parasites have to trespass many barriers to successfully settle within their mammalian host, which is equipped with mechanical borders and complex weaponry of an evolved immune system. However, little is known about mechanisms of early local events in filarial infections. In this study, bone marrow-derived dendritic cells not only upregulated activation markers CD40 and CD80 upon in vitro stimulation with filarial extracts, but also secreted CCL17, a chemokine known to be produced upon microbial challenge. Mice deficient for CCL17 had an up to 4-fold higher worm burden compared with controls by day 10 of infection with the murine filaria Litomosoides sigmodontis. Also, numbers of mast cells (MCs) invading the skin and degranulation were significantly increased, which was associated with enhanced vascular permeability and larval establishment. This phenotype was reverted by inhibition of MC degranulation with disodium cromoglycate or by blockade of histamine. In addition, we showed that CCL17-mediated vascular permeability was dependent on the presence of Wolbachia endosymbionts and TLR2. Our findings reveal that CCL17 controls filarial larval entry by limiting MC-dependent vascular permeability.