An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.

The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

Self-recognition through Dectin-1 exacerbates liver inflammation.

Dectin-1 is a well-characterized C-type lectin receptor involved in anti-fungal immunity through the recognition of polysaccharides; however, molecular mechanisms and outcomes initiated through self-recognition have not been fully understood. Here, we purified a water-soluble fraction from mouse liver that acts as a Dectin-1 agonist. To address the physiological relevance of this recognition, we utilized sterile liver inflammation models. The CCl4-induced hepatitis model showed that Dectin-1 deficiency led to reduced inflammation through decreased inflammatory cell infiltration and lower pro-inflammatory cytokine levels. Moreover, in a NASH model induced by streptozotocin and a high-fat diet, hepatic inflammation and fibrosis were ameliorated in Dectin-1-deficient mice. The Dectin-1 agonist activity was increased in the water-soluble fraction from NASH mice, suggesting a potential pathogenic cycle between Dectin-1 activation and hepatitis progression. In vivo administration of the fraction into mice induced hepatic inflammation. These results highlight a role of self-recognition through Dectin-1 that triggers hepatic innate immune responses and contributes to the exacerbation of inflammation in pathogenic settings. Thus, the blockade of this axis may provide a therapeutic option for liver inflammatory diseases.

C. albicans N-linked mannans potentiate the induction of trained immunity via Dectin-2.

The interaction between the Candida albicans cell wall and pattern recognition receptors is crucial for the initiation of host immune responses which, ultimately, contribute to the clearance of this pathogenic fungus. In the present study, we investigate the ability of C. albicans mannans to modulate immune response and induce innate immune memory (also termed trained immunity). Using mutants of C. albicans that are defective in, or lack mannosyl residues, we show that alterations in the mannosylation of the C. albicans cell wall affect the innate cytokine response and strongly reduce the secretion of T cell-derived cytokines. Subsequently, we demonstrate that the branching of N-linked mannan, but not O-linked mannan, is essential to potentiate the induction of trained immunity, a process mediated by Dectin-2. In conclusion, N-linked mannan is needed, in addition to ß-glucans, for an effective induction of trained immunity by C. albicans.

Cholinergic signaling via the α7 nicotinic acetylcholine receptor regulates the migration of monocyte-derived macrophages during acute inflammation.

BACKGROUND: The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. RESULTS: We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, ß1 and ß2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMß2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. CONCLUSIONS: We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

Innate Immune Memory and the Host Response to Infection.

Unlike the adaptive immune system, the innate immune system has classically been characterized as being devoid of memory functions. However, recent research shows that innate myeloid and lymphoid cells have the ability to retain memory of prior pathogen exposure and become primed to elicit a robust, broad-spectrum response to subsequent infection. This phenomenon has been termed innate immune memory or trained immunity. Innate immune memory is induced via activation of pattern recognition receptors and the actions of cytokines on hematopoietic progenitors and stem cells in bone marrow and innate leukocytes in the periphery. The trained phenotype is induced and sustained via epigenetic modifications that reprogram transcriptional patterns and metabolism. These modifications augment antimicrobial functions, such as leukocyte expansion, chemotaxis, phagocytosis, and microbial killing, to facilitate an augmented host response to infection. Alternatively, innate immune memory may contribute to the pathogenesis of chronic diseases, such as atherosclerosis and Alzheimer's disease.

Descriptive Analysis of Transient-State Observations for Thioredoxin/Glutathione Reductase (Sec597Cys) from Schistosoma mansoni.

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both oxidized thioredoxin and glutathione with electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH). SmTGR is a drug target for the treatment of Schistosomiasis, an infection caused by Schistosoma platyhelminths residing in the blood vessels of the host. Schistosoma spp. are reliant on TGR enzymes as they lack catalase and so use reduced thioredoxin and glutathione to regenerate peroxiredoxins consumed in the detoxification of reactive oxygen species. SmTGR is a flavin adenine dinucleotide (FAD)-dependent enzyme, and we have used the flavin as a spectrophotometric reporter to observe the movement of electrons within the enzyme. The data show that NADPH fractionally reduces the active site flavin with an observed rate constant estimated in this study to be ∼3000 s-1. The flavin then reoxidizes by passing electrons at a similar rate to the proximal Cys159-Cys154 disulfide pair. The dissociation of NADP+ occurs with a rate of ∼180 s-1, which induces the deprotonation of Cys159, and this coincides with the accumulation of an intense FAD-thiolate charge transfer band. It is proposed that the electrons then pass to the Cys596-Cys597 disulfide pair of the associated subunit in the dimer with a net rate constant of ∼2 s-1. (Note: Cys597 is Sec597 in wild-type (WT) SmTGR.) From this position, the electrons can be passed to oxidized thioredoxin or further into the protein to reduce the Cys28-Cys31 disulfide pair of the originating subunit of the dimer. From the Cys28-Cys31 center, electrons can then pass to oxidized glutathione that has a binding site directly adjacent.

ß-Glucan Induces Distinct and Protective Innate Immune Memory in Differentiated Macrophages.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. ß-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that ß-glucan protects mice from Pseudomonas aeruginosa infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using ß-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow-derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. ß-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic ß-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that ß-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

Traditional Kenyan herbal medicine: exploring natural products' therapeutics against schistosomiasis.

Praziquantel (PZQ) remains the only drug of choice for the treatment of schistosomiasis, caused by parasitic flatworms. The widespread use of PZQ in schistosomiasis endemic areas for about four decades raises concerns about the emergence of resistance of Schistosoma spp. to PZQ under drug selection pressure. This reinforces the urgency in finding alternative therapeutic options that could replace or complement PZQ. We explored the potential of medicinal plants commonly used by indigenes in Kenya for the treatment of various ailments including malaria, pneumonia, and diarrhoea for their antischistosomal properties. Employing the Soxhlet extraction method with different solvents, seven medicinal plants Artemisia annua, Ajuga remota, Bredilia micranta, Cordia africana, Physalis peruviana, Prunus africana and Senna didymobotrya were extracted. Qualitative phytochemical screening was performed to determine the presence of various phytochemicals in the plant extracts. Extracts were tested against Schistosoma mansoni newly transformed schistosomula (NTS) and adult worms and the schistosomicidal activity was determined by using the adenosine triphosphate quantitation assay. Phytochemical analysis of the extracts showed different classes of compounds such as alkaloids, tannins, terpenes, etc., in plant extracts active against S. mansoni worms. Seven extracts out of 22 resulted in <20% viability against NTS in 24 h at 100 µg/ml. Five of the extracts with inhibitory activity against NTS showed >69.7% and ≥72.4% reduction in viability against adult worms after exposure for 24 and 48 h, respectively. This study provides encouraging preliminary evidence that extracts of Kenyan medicinal plants deserve further study as potential alternative therapeutics that may form the basis for the development of the new treatments for schistosomiasis.

Taking Advantage of the Morpheein Behavior of Peroxiredoxin in Bionanotechnology.

Morpheeins are proteins that reversibly assemble into different oligomers, whose architectures are governed by conformational changes of the subunits. This property could be utilized in bionanotechnology where the building of nanometric and new high-ordered structures is required. By capitalizing on the adaptability of morpheeins to create patterned structures and exploiting their inborn affinity toward inorganic and living matter, "bottom-up" creation of nanostructures could be achieved using a single protein building block, which may be useful as such or as scaffolds for more complex materials. Peroxiredoxins represent the paradigm of a morpheein that can be applied to bionanotechnology. This review describes the structural and functional transitions that peroxiredoxins undergo to form high-order oligomers, e.g., rings, tubes, particles, and catenanes, and reports on the chemical and genetic engineering approaches to employ them in the generation of responsive nanostructures and nanodevices. The usefulness of the morpheeins' behavior is emphasized, supporting their use in future applications.

Multimodality characteristics of multifocal choroid plexus carcinoma with bilateral calvarial defects in a dog.

An 8-year-old male intact miniature poodle presented for blindness, obtundation, tetraparesis, and vestibular signs. Magnetic resonance imaging, radiography, and ultrasound revealed a left piriform lobe lesion, right cerebellar and left brainstem lesions, and hydrocephalus and bilateral calvarial defects. Histopathology confirmed a choroid plexus carcinoma with meningeal and intraventricular metastases. The calvarial defect did not show evidence of necrosis, osteoclastic resorption, inflammation or neoplastic infiltration, reflecting a quiescent calvarial atrophy or dysplasia. These novel findings supported inclusion of bone atrophy secondary to chronic increased intracranial pressure as a differential diagnosis for large calvarial defects in dogs with choroid plexus carcinoma.

Engineering Nanogels for Drug Delivery to Pathogenic Fungi Aspergillus fumigatus by Tuning Polymer Amphiphilicity.

Invasive aspergillosis is a serious threat to immunodeficient and critically ill patients caused mainly by the fungus Aspergillus fumigatus. Here, poly(glycidol)-based nanogels (NGs) are proposed as delivery vehicles for antifungal agents for sustained drug release. NGs are formed by simple self-assembly of random copolymers, followed by oxidative cross-linking of thiol functionalities. We investigate the impact of copolymer amphiphilicity on NG interaction with mature fungal hyphae in order to select the optimal drug delivery system for model antifungal drug amphotericin B. The results show that drug-loaded NGs decrease minimal inhibitory concentration (MIC) for around four times and slow down the fungal biofilm synthesis at concentrations lower than MIC. Our results suggest that amphiphilicity of nanoparticle's polymer matrix is an important factor in understanding the action of nanocarriers toward fungal cells and should be considered in the development of nanoparticle-based antifungal therapy.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions.

ß-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of ß-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma ß-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and ß-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of ß-glucan and the results confirmed that ß-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Immunoregulatory Activity of the Natural Product Laminarin Varies Widely as a Result of Its Physical Properties.

Ligation of Dectin-1 by fungal glucans elicits a Th17 response that is necessary for clearing many fungal pathogens. Laminarin is a (1→3, 1→6)-ß-glucan that is widely reported to be a Dectin-1 antagonist, however, there are reports that laminarin is also a Dectin-1 agonist. To address this controversy, we assessed the physical properties, structure, purity, Dectin-1 binding, and biological activity of five different laminarin preparations from three different commercial sources. The proton nuclear magnetic resonance analysis indicated that all of the preparations contained laminarin although their molecular mass varied considerably (4400-34,400 Da). Two of the laminarins contained substantial quantities of very low m.w. compounds, some of which were not laminarin. These low m.w. moieties could be significantly reduced by extensive dialysis. All of the laminarin preparations were bound by recombinant human Dectin-1 and mouse Dectin-1, but the affinity varied considerably, and binding affinity did not correlate with Dectin-1 agonism, antagonism, or potency. In both human and mouse cells, two laminarins were Dectin-1 antagonists and two were Dectin-1 agonists. The remaining laminarin was a Dectin-1 antagonist, but when the low m.w. moieties were removed, it became an agonist. We were able to identify a laminarin that is a Dectin-1 agonist and a laminarin that is Dectin-1 antagonist, both of which are relatively pure preparations. These laminarins may be useful in elucidating the structure and activity relationships of glucan/Dectin-1 interactions. Our data demonstrate that laminarin can be either a Dectin-1 antagonist or agonist, depending on the physicochemical properties, purity, and structure of the laminarin preparation employed.

The TLR4 Agonist Monophosphoryl Lipid A Drives Broad Resistance to Infection via Dynamic Reprogramming of Macrophage Metabolism.

Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with Staphylococcus aureus and Candida albicans that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.

Repurposing Auranofin and Evaluation of a New Gold(I) Compound for the Search of Treatment of Human and Cattle Parasitic Diseases: From Protozoa to Helminth Infections.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

FACTORS AFFECTING TEAR PRODUCTION AND INTRAOCULAR PRESSURE IN ANESTHETIZED CHIMPANZEES (PAN TROGLODYTES).

Measurements of intraocular pressure (IOP) and tear production are key components of ophthalmic examination. Chimpanzees (Pan troglodytes) were anesthetized using either tiletamine-zolazepam (TZ; 2 mg/kg) combined with medetomidine (TZM; 0.02 mg/kg), or, TZ alone (6mg/kg). Tear production was lower (P = 0.03) with TZM (5.63 ± 6.22 mm/min; n = 16) than with TZ (11.13 ± 4.63 mm/min; n = 8). Mean IOP, measured using rebound tonometry in an upright body position (n = 8) was 18.74 ± 3.01 mm Hg, with no differences between right and left eyes. However, positioning chimpanzees in left lateral recumbency (n = 27) resulted in higher IOP in the dependent (left) eye (24.77 ± 4.49 mm Hg) compared to the nondependent (right) eye (22.27 ± 4.65 mm Hg) of the same animal (P < 0.0001). These data indicate medetomidine anesthesia markedly lowers tear production in chimpanzees, and that body position should be taken into consideration when performing rebound tonometry.