Stimulation Of Innate Immune Cells Research Articles

The impact of trained immunity in respiratory viral infections.

Epidemic peaks of respiratory viruses that co-circulate during the winter-spring seasons can be synchronous or asynchronous. The occurrence of temporal patterns in epidemics caused by some respiratory viruses suggests that they could negatively interact with each other. These negative interactions may result from a programme of innate immune memory, known as trained immunity, which may confer broad protective effects against respiratory viruses. It is suggested that stimulation of innate immune cells by a vaccine or a pathogen could induce their long-term functional reprogramming through an interplay between metabolic and epigenetic changes, which influence the transcriptional response to a secondary challenge. During the coronavirus disease 2019 pandemic, the circulation of most respiratory viruses was prevented by non-pharmacological interventions and then resumed at unusual periods once sanitary measures were lifted. With time, respiratory viruses should find again their own ecological niches. This transition period provides an opportunity to study the interactions between respiratory viruses at the population level.

Self-replicating RNA nanoparticle vaccine elicits protective immune responses against SARS-CoV-2

The creation of safe and effective vaccines that induce potent cellular and humoral immune responses against SARS-CoV-2 is urgently needed to end the global COVID-19 epidemic. Here, we developed an alphavirus-derived self-replicating RNA (repRNA)-based vaccine platform encoding the receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein. The repRNA triggers prolonged antigen expression compared with conventional mRNA due to the replication machinery of repRNA. To improve the delivery and vaccine efficacy of repRNA, we developed a self-assembling liposome-protamine-RNA (LPR) nanoparticle with highly efficient encapsulation and transfection of repRNA. LPR-repRNA vaccines substantially activated type I interferon response and innate immune signaling pathways. Subcutaneous immunization of LPR-repRNA-RBD led to prolonged antigen expression, stimulation of innate immune cells, and induction of germinal center response in draining lymph nodes. LPR-repRNA-RBD induced antigen-specific Tcell responses and skewed cellular immunity toward an effector memory CD8+ Tcell response. Immunizations with LPR-repRNA-RBD triggered the production of anti-RBD IgG antibodies and induced neutralizing antibody response against SARS-CoV-2 pseudovirus. LPR-repRNA-RBD vaccines reduced SARS-CoV-2 infection and lung inflammation in mice. Altogether, these data suggest that the LPR-repRNA platform can be a promising avenue for COVID-19 vaccine development.

Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses.

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of "first responders" (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and Tcell responses invivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.

Cutting both ways: the innate immune response to oncolytic virotherapy.

Oncolytic viruses (OVs), above and beyond infecting and lysing malignant cells, interact with the immune system in complex ways that have important therapeutic significance. While investigation into these interactions is still in its early stages, important insights have been made over the past two decades that will help improve the clinical efficacy of OV-based management strategies in cancer care moving forward. The inherent immunosuppression that defines the tumor microenvironment can be modified by OV infection, and the subsequent recruitment and activation of innate immune cells, in particular, is central to this. Indeed, neutrophils, macrophages, natural killer cells, and dendritic cells, as well as other populations such as myeloid-derived suppressor cells, are key to the immune escape that allows tumors to survive, but their natural response to infection can be exploited by virotherapy. While stimulation of innate immune cells by OVs can initiate antitumor responses, related antiviral activity can limit virus spread and direct cytopathogenic effects. In this review, we highlight how each innate immune cell population influences this balance of antitumor and antiviral forces during virotherapy, some of the important molecular pathways that have been identified, and specific therapeutic targets that have emerged through this work. We discuss the importance of OV-based combination therapies in optimizing antiviral and antitumor innate immune responses stimulated by virotherapy toward tumor eradication, and how these processes vary depending on the tumor and OV in question. Rather than concentrating on a particular OV species in the review, we present the range of effects that have been documented across OV types to emphasize the context-specific nature of these interactions and how this is important in the design of future OV-based treatment approaches.

Therapeutic Synergy Between Antibiotics and Pulmonary Toll-Like Receptor 5 Stimulation in Antibiotic-Sensitive or -Resistant Pneumonia.

Bacterial infections of the respiratory tract constitute a major cause of death worldwide. Given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. In this context, innovative therapeutic strategies are urgently needed. Stimulation of innate immune cells in the respiratory tract [via activation of Toll-like receptors (TLRs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. Previous studies of the TLR5 agonist flagellin in animal models showed that standalone TLR stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. Here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. Using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic–susceptible pneumococcus. We next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. Remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than 100-fold compared to standalone treatments. Our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. In conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria.

Expression kinetics of ISG15, IRF3, IFNγ, IL10, IL2 and IL4 genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in PPRV vaccinated, challenged, infected sheep and goats

Here, we studied the in vivo expression of Th1 (IL2 and IFN gamma) and Th2 (IL4 and IL10) - cytokines and antiviral molecules - IRF3 and ISG15 in peripheral blood mononuclear cells in relation to antigen and antibody dynamics under Peste des petits ruminants virus (PPRV) vaccination, infection and challenge in both sheep and goats. Vaccinated goats were seropositive by 9 days post vaccination (dpv) while in sheep idiosyncratic response was observed between 9 and 14 dpv for different animals. Expression of PPRV N gene was not detected in PBMCs of vaccinated and vaccinated challenged groups of both species, but was detected in unvaccinated infected PBMCs at 9 and 14 days post infection. The higher viral load at 9 dpi coincided with the peak clinical signs of the disease. The peak in viral replication at 9 dpi correlated with significant expression of antiviral molecules IRF3, ISG15 and IFN gamma in both the species. With the progression of disease, the decrease in N gene expression also correlated with the decrease in expression of IRF3, ISG15 and IFN gamma. In the unvaccinated infected animals ISG15, IRF3, IFN gamma and IL10 expression was higher than vaccinated animals. The IFN gamma expression predominated over IL4 in both vaccinated and infected animals with the infected exhibiting a stronger Th1 response. The persistent upregulation of this antiviral molecular signature - ISG15 and IRF3 even after 2 weeks post vaccination, presumably reflects the ongoing stimulation of innate immune cells.

Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection

Therapeutic vaccine research and development are especially important in Chagas disease considering the characteristics of the chronic infection and the number of people in the Americas living with a parasite infection for decades. We have previously reported the efficacy of attenuated Salmonella enterica (S) carrying plasmid encoding cruzipain (SCz) to protect against Trypanosoma cruzi infection. In the present work we investigated whether Cz DNA vaccine immunotherapy could be effective in controlling an ongoing T. cruzi infection in mice. We here report the intramuscular administration of naked Cz DNA or the oral administration of Salmonella as Cz DNA delivery system as therapeutic vaccines in mice during acute or chronic infection. The coadministration of a plasmid encoding GM-CSF improved vaccine performance, indicating that the stimulation of innate immune cells is needed in the event of an ongoing infection. These therapeutic vaccines were able to address the response to a protective and sustained Th1 biased profile not only against Cz but also against a variety of parasite antigens. The combined therapeutic vaccine during the chronic phase of infection prevents tissue pathology as shown by a reduced level of enzyme activity characteristic of tissue damage and a tissue status compatible with normal tissue. The obtained results suggest that immunotherapy with Cz and GM-CSF DNAs, either alone or in combination with other drug treatments, may represent a promising alternative for Chagas disease therapy.

Andosan?-An Anti-Allergic and Anti-Inflammatory Ingredient Prepared from Agaricus blazei Mushroom

Andosan™ is an extract prepared from medicinal Basidiomycetes mushrooms, mainly Agaricus blazei Murill (AbM) (82%), but it also contains Hericeum erinaceus (He) (15%) and Grifola frondosa (Gf) (3%), all of which have immunomodulating properties. Whereas He and Gf have been used in traditional Chinese and Japanese medicine against cancer, AbM originates from Brazilian rain forest and was used locally and later abroad as health food for the prevention of a range of diseases, including chronic hepatitis, diabetes and cancer. The mushrooms’ health effects are probably due to their content of A¯ÂÂ¢-glucans and other smaller immunomodulating polysaccharides and proteoglucans. These effects are mediated through their stimulation of innate immune cells, such as monocytes, NK cells and dendritic cells, and the amelioration of a skewded Th1/Th2 balance and of inflammation. Here, we give an overview of the anti-allergic effects of Andosan™ in a mouse model for allergy and anti-inflammatory effects both in vitro, in healthy individuals and in patients with inflammatory bowel diseases.

Stimulation of Innate Immune Cells Induced by Probiotics: Participation of Toll-Like Receptors

Objective: The present work aimed to study the functionality of macrophages from different locations (peritoneum, spleen and Peyer´s patches) when they were stimulated with probiotics microorganisms: Lactobacillus casei CRL 431 and Lactobacillus paracasei CNCM I-1518 or a Probiotic Fermented Milk (PFM) through Toll-Like Receptors (TLRs), prior challenged with agonists or antagonists of TLRs. Methods: BALB/c mice received in the drinking water, the probiotic bacteria (L. casei CRL 431 and L. paracasei CNCM I-1518) or the PFM. We focused our investigation mainly on the phagocytic activity of macrophages from peritoneum, spleen and Peyer’s patches and cytokine production were evaluated prior challenged with TLR2 and TLR4 agonists or antagonists. The microbicidal activity of macrophages and against an infection with Salmonella typhimurium was also studied. To assess the role of TLR in probiotic stimulation, we evaluated the phagocytic activity, cytokine production and Immunoglobin G (IgG) anti-OVA in C57BL/6 knockout mice to MyD88, TLR2 and TLR4. Results: In BALB/c mice, the best effect in the phagocytosis assay was obtained with the probiotic bacteria L. casei CRL 431, this effect was reinforced with TLR2 agonist. The production of different cytokines (IL-10 and IL-6) was improved with the probiotic treatments and this production was ameliorated with TLRs agonists. The antimicrobial activity was increased with L. casei CRL 431 and L. paracasei CNCM I-1518, TLR2 and TLR4 antagonists had a negative effect on microbicidal activity. The administration of probiotic bacteria or PFM improved the host response against S. typhimurium controlling the infection during the first hours post-infection. In C57BL/6 knockout mice, phagocytic activity was significantly diminished in comparison to wild type mice and the probiotic bacteria or PFM administration was not able to improve this activity. The IL-10 production was increased at a concentration of 108 cells/ml of L. casei CRL 431 in TLR2-/- and TLR4-/-, but not in MyD88-/- mice. The administration of probiotic bacteria or PFM did not play a stimulating effect in the systemic immune response against to OVA antigen in knockout mice. Conclusions: Probiotics modulate the different signaling pathways of innate immune cells through the TLRs. The macrophages activation depends on location of them and that different probiotic strains of Lactobacilli can evoke different intensity of responses. The data suggest that probiotic not only promote a major expression of TLRs but also use these receptors via the innate immune cells as macrophages to stimulate and modulate the immune response.

Stimulation of innate immune cells by light-activated TLR7/8 agonists.

The innate immune response is controlled, in part, by the synergistic interaction of multiple Toll-like receptors (TLRs). This multi-receptor cooperation is responsible for the potent activity of many vaccines, but few tools have been developed to understand the spatio-temporal elements of TLR synergies. In this Communication, we present photo-controlled agonists of TLR7/8. By strategically protecting the active agonist moiety based on an agonist-bound crystal structure, TLR activity is suppressed and then regained upon exposure to light. We confirmed NF-κB production upon light exposure in a model macrophage cell line. Primary cell activity was confirmed by examining cytokine and cell surface marker production in bone-marrow-derived dendritic cells. Finally, we used light to activate dendritic cell sub-populations within a larger population.

Nanolipoprotein delivery enhances immunostimulatory properties of innate immune agonists and provides protection against lethal influenza challenge (P4218)

Abstract Therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms. As such, we have employed a nanolipoprotein (NLP) platform to enhance the efficacy of synthetic innate immune agonists. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. We demonstrate that the synthetic Toll-like receptor (TLR) ligands monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. Further, conjugation of both MPLA and CpG ODNs to NLPs significantly enhances their immunostimulatory profiles in vivo compared to administration of agonists alone. Specifically, conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, enhances and prolongs stimulation of innate immune cells, significantly increases cytokine production, and enhances upregulation of myriad immunoregulatory genes in vivo. Importantly, enhancement of cytokine production by agonists conjugated to NLPs was also observed in primary human dendritic cells. Strikingly, BALB/c mice pretreated with NLP-CpG constructs prior to a lethal influenza challenge were protected from weight-loss induced death, whereas pretreatment with CpG alone had no effect on survival. Taken together, these data suggest that NLP:agonist conjugates potentially represent a novel therapeutic measure against emerging pathogens.

Composition of Herba Pogostemonis Water Extract and Protection of Infected Mice against Salmonella Typhimurium-Induced Liver Damage and Mortality by Stimulation of Innate Immune Cells

GC-MS analysis of a hot water extract of Herba Pogostemonis (HP) revealed the presence of 131 compounds. HP slightly inhibited Salmonella Typhimurium bacteria in culture and stimulated uptake of the bacteria into RAW 264.7 murine macrophage cells as indicated by both increased fluorescence from internalized FITC-dextran and increased colony-forming unit (CFU) counts of the lysed macrophages. Postinfection, the HP-treated cells showed lower bacterial counts than the control. HP elicited altered morphology, elevated inducible NO synthase (iNOS) mRNA, and reduced pro-inflammatory cytokine expression in macrophage cells. Salmonella induced increased expression of iNOS mRNA, cognate polypeptides, and NO. Histology of mice infected with a sublethal dose (1 × 10(4) CFU) of Salmonella showed that intraperitoneally administered HP protected against necrosis of the liver, a biomarker of in vivo salmonellosis. The lifespan of mice infected with a lethal dose (1 × 10(5) CFU) was significantly extended. These results suggest that the activity of HP against bacterial infection in mice occurs through the activation of innate immune macrophage cells. The relationship of composition of HP to bioactivity is discussed.

A Cluster of Interferon-γ-Inducible p65 GTPases Plays a Critical Role in Host Defense against Toxoplasma gondii

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T.gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T.gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T.gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T.gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Hericium erinaceus Mushroom Extracts Protect Infected Mice against Salmonella Typhimurium-Induced Liver Damage and Mortality by Stimulation of Innate Immune Cells

The present study investigated the antibacterial effect of four extracts from the fruitbody of the edible medicinal mushroom Hericium erinaceus (hot water extract, HWE; microwave/50% ethanol extract, MWE; acid extract, ACE; and alkaline extract, AKE) against murine salmonellosis. The extracts had no effect on Salmonella ser. Typhimurium growth in culture. Nor were the extracts toxic to murine macrophage cells, RAW 264.7. HWE and MWE stimulated uptake of the bacteria into the macrophage cells as indicated by increased colony-forming unit (CFU) counts of the contents of the lysed macrophages infected with Salmonella Typhimurium for 30 and 60 min. Two hours postinfection, the bacterial counts increased in the macrophages, but 4 and 8 h postinfection the HWE- and MWE-treated cells showed greater activity against the bacteria than the control. HWE- and MWE-treated noninfected macrophages had altered morphology and elevated inducible nitric oxide (NO) synthase (iNOS) mRNA expression. In the presence of S. Typhimurium, iNOS mRNA expression was further increased, accompanied by an increase in NO production. Histology assays of the livers of mice infected with a sublethal dose (1 × 10(4) CFU) of S. Typhimurium showed that HWE and MWE, administered by daily intraperitoneal injection, protected against necrosis of the liver, a biomarker of in vivo salmonellosis. The lifespans of mice similarly infected with a lethal dose of S. Typhimurium (1 × 10(5) CFU) were significantly extended by HWE and MWE. β-Glucan, known to stimulate the immune system, was previously found to be present in high amounts in the active extracts. These results suggest that the mushroom extract activities against bacterial infection in mice occur through the activation of innate immune cells.

The MushroomAgaricus blazeiMurill Elicits Medicinal Effects on Tumor, Infection, Allergy, and Inflammation through Its Modulation of Innate Immunity and Amelioration of Th1/Th2 Imbalance and Inflammation

The medicinal mushroom Agaricus blazei Murill from the Brazilian rain forest has been used in traditional medicine and as health food for the prevention of a range of diseases, including infection, allergy, and cancer. Other scientists and we have examined whether there is scientific evidence behind such postulations. Agaricus blazei M is rich in the immunomodulating polysaccharides, β-glucans, and has been shown to have antitumor, anti-infection, and antiallergic/-asthmatic properties in mouse models, in addition to anti-inflammatory effects in inflammatory bowel disease patients. These effects are mediated through the mushroom's stimulation of innate immune cells, such as monocytes, NK cells, and dendritic cells, and the amelioration of a skewed Th1/Th2 balance and inflammation.

Microbiota

We have recently demonstrated that microbiota stimulation of innate immune pathways is required for T cell spontaneous proliferation and chronic intestinal inflammation. Microbiota promoted spontaneous proliferation of T cells by activating dendritic cells to produce interleukin (IL)-6 via a TLR/MyD88-dependent pathway. Although both CBir1-specific TCR transgenic (CBir1 Tg) T cells, which are specific for an immunodominant microbiota antigen, and OT-II T cells, which are specific for the model antigen ovalbumin, underwent spontaneous proliferation, only CBir1 Tg T cells but not OT-II T cells induced colitis in specific pathogen-free RAG-/- mice. Blockade of IL-6 or IL-6-mediated spontaneous proliferation of CBir1 Tg T cells abrogated colitis induction in this adoptive transfer model. Our data reveal that microbiota serves as a natural adjuvant for T cell spontaneous proliferation and development of chronic intestinal inflammation, and that both microbiota stimulation of innate immune cells with subsequent T cell spontaneous proliferation and microbiota antigen activation of antigen-specific TCR are required for the induction of experimental colitis

Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogrammingMycobacterium tuberculosis‐infected macrophages

Wnt/Frizzled signaling, essential for embryonic development, has also recently been implicated in the modulation of inflammatory processes. In the current study, we observed a reciprocal regulation of the Toll-like receptor (TLR)/nuclear factor-κB (NF-κB) and the Wnt/β-catenin pathway after aerosol infection of mice with Mycobacterium tuberculosis: whereas proinflammatory mediators were substantially increased, β-catenin signaling was significantly reduced. A systematic screen of Fzd homologs in infected mice identified Fzd1 mRNA to be significantly up-regulated during the course of infection. In vitro infection of murine macrophages led to a strong induction of Fzd1 that was dependent on TLRs, the myeloid differentiation response gene 88 (MyD88), and a functional NF-κB pathway. Flow cytometry demonstrated an elevated Fzd1 expression on macrophages in response to M. tuberculosis that was synergistically enhanced in the presence of IFN-γ. Addition of the Fzd1 ligand Wnt3a induced Wnt/β-catenin signaling in murine macrophages that was inhibited in the presence of a soluble Fzd1/Fc fusion protein. Furthermore, Wnt3a reduced TNF release, suggesting that Wnt3a promotes anti-inflammatory functions in murine macrophages. The current data support the notion that evolutionarily conserved Wnt/Fzd signaling is involved in balancing the inflammatory response to microbial stimulation of innate immune cells of vertebrate origin.

Wnt proteins differenzially regulate macrophage functions in the context of M. tuberculosis infection

Wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. They are best known for their role in embryonic development and tissue homeostasis. We have recently identified Wnt5a and Frizzled5 to be functionally involved in the regulation of the Th1 cytokine response indicating a role in antimicrobial defense (Blumenthal et al, Blood 2006). In a systematic screen of Wnt/Fzd homologs in murine bone marrow derived macrophages, we have now identified Frizzled1 to be upregulated in response to mycobacteria and conserved bacterial structures. We show that increased Fzd1 transcription was dependent on TLR2, MyD88, NF-kappaB and TNF-alpha release. Macrophage Fzd1 expression was confirmed on protein level by Western blot and on a single cell level by FACS analysis. Infection of bone marrow derived macrophages with M. tuberculosis led to enhanced Fzd1 surface expression. In lung homogenates of M. tuberculosis infected mice Fzd1 mRNA was shown to be upregulated, whereas transcripts of its putative ligand Wnt3a were found constitutively expressed. By immunohistochemistry the localisation of Fzd1 and Wnt3a expressing cells was addressed. Functional studies revealed that Wnt3a-induced Wnt/beta-catenin signaling in macrophages was inhibited by a soluble Fzd1 fusion protein. Activation of Wnt/beta-catenin signaling modulated the immune response of macrophages to mycobacterial infection. Both Toll and Wnt signaling pathways are evolutionarily highly conserved and have only recently been found to intersect in Drosophila. The current data further support the notion that Wnt/Fzd signaling is involved in orchestrating the immune response due to microbial stimulation of innate immune cells.

IFN-β Production by TLR4-Stimulated Innate Immune Cells Is Negatively Regulated by GSK3-β

TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-beta. In this study, we demonstrate glycogen synthase kinase 3-beta (GSK3-beta) plays a fundamental role in this process. Suppression of GSK3-beta activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-beta mutant enhanced IFN-beta production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-beta mutant severely attenuated IFN-beta production. GSK3-beta was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-beta inhibition to augment IFN-beta, demonstrating that the ability of GSK3 to control IFN-beta production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-beta production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-beta upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-beta production by TLR4-stimulated innate immune cells.

CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells

Cells of the innate immune system play important roles in the progression of prion disease after peripheral infection. It has been found in vivo and in vitro that the expression of the cellular prion protein (PrP(c)) is up-regulated on stimulation of immune cells, also indicating the functional importance of PrP(c) in the immune system. The aim of our study was to investigate the impact of cytosine-phosphate-guanosine- and lipopolysaccharide-induced PrP(c) up-regulation on the uptake and processing of the pathological prion protein (PrP(Sc)) in phagocytic innate immune cells. For this purpose, we challenged the macrophage cell line J774, the microglial cell line BV-2 and primary bone marrow-derived macrophages in a resting or stimulated state with various prion strains, and monitored the uptake and clearance of PrP(Sc). Interestingly, stimulation led either to a transient increase in the level of PrP(Sc) relative to unstimulated cells or to a decelerated degradation of PrP(Sc). These features were dependent on cell type and prion strain. Our data indicate that the stimulation of innate immune cells may be able to support transient prion propagation, possibly explained by an increased PrP(c) cell surface expression in stimulated cells. We suggest that stimulation of innate immune cells can lead to an imbalance between the propagation and degradation of PrP(Sc).