c-Met+ Cytotoxic T Lymphocytes Exhibit Enhanced Cytotoxicity in Mice and Humans In Vitro Tumor Models.

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell-cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met- CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met- CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer.

Variations in Biodistribution and Acute Response of Differently Shaped Titania Nanoparticles in Healthy Rodents.

Titanium dioxide nanoparticles (TiO2 NPs) are one of the main sources of the nanoparticulate matter exposure to humans. Although several studies have demonstrated their potential toxic effects, the real nature of the correlation between NP properties and their interaction with biological targets is still far from being fully elucidated. Here, engineered TiO2 NPs with various geometries (bipyramids, plates, and rods) have been prepared, characterized and intravenously administered in healthy mice. Parameters such as biodistribution, accumulation, and toxicity have been assessed in the lungs and liver. Our data show that the organ accumulation of TiO2 NPs, measured by ICP-MS, is quite low, and this is only partially and transiently affected by the NP geometries. The long-lasting permanence is exclusively restricted to the lungs. Here, bipyramids and plates show a higher accumulation, and interestingly, rod-shaped NPs are the most toxic, leading to histopathological pulmonary alterations. In addition, they are also able to induce a transient increase in serum markers related to hepatocellular injury. These results indicate that rods, more than bipyramidal and spherical geometries, lead to a stronger and more severe biological effect. Overall, small physico-chemical differences can dramatically modify both accumulation and safety.

Continuous sensing of IFNα by hepatic endothelial cells shapes a vascular antimetastatic barrier.

Hepatic metastases are a poor prognostic factor of colorectal carcinoma (CRC) and new strategies to reduce the risk of liver CRC colonization are highly needed. Herein, we used mouse models of hepatic metastatization to demonstrate that the continuous infusion of therapeutic doses of interferon-alpha (IFNα) controls CRC invasion by acting on hepatic endothelial cells (HECs). Mechanistically, IFNα promoted the development of a vascular antimetastatic niche characterized by liver sinusoidal endothelial cells (LSECs) defenestration extracellular matrix and glycocalyx deposition, thus strengthening the liver vascular barrier impairing CRC trans-sinusoidal migration, without requiring a direct action on tumor cells, hepatic stellate cells, hepatocytes, or liver dendritic cells (DCs), Kupffer cells (KCs) and liver capsular macrophages (LCMs). Moreover, IFNα endowed LSECs with efficient cross-priming potential that, along with the early intravascular tumor burden reduction, supported the generation of antitumor CD8+ T cells and ultimately led to the establishment of a protective long-term memory T cell response. These findings provide a rationale for the use of continuous IFNα therapy in perioperative settings to reduce CRC metastatic spreading to the liver.

Colorectal cancer remains one of the most widespread and deadly cancers worldwide. Poor health outcomes are usually linked to diseased cells spreading from the intestine to create new tumors in the liver or other parts of the body. Treatment involves surgically removing the initial tumors in the bowel, but patient survival could be improved if, in parallel, their immune system was 'boosted' to destroy cancer cells before they can form other tumors. Interferon alpha is a small protein which helps to coordinate how the immune system recognizes and deactivates foreign agents and cancerous cells. It has recently been trialed as a colorectal cancer treatment to prevent tumors from spreading to the liver, but only with limited success. This partly because interferon-alpha is usually administered in high and pulsed doses, which cause severe side effects through the body. Instead, Tran, Ferreira, Alvarez-Moya et al. aimed to investigate whether continuously delivering lower amounts of the drug could be a better approach. This strategy was tested on mice in which colorectal cancer cells had been implanted into the wall of the large intestine. Continuous administration minimized the risk of the implanted cancer cells spreading to the liver while also creating fewer side effects. The team was able to identify an optimum delivery strategy by varying how much interferon-alpha the animals received and when. Further experiments also revealed a new mechanism by which interferon-alpha prevented the spread of colorectal cancer. Upon receiving continuous doses of the drug, a group of liver cells started to generate a physical barrier which stopped cancer cells from being able to invade the organ. The treatment also promoted long-term immune responses that targeted diseased cells while being safe for healthy tissues. If confirmed in clinical trials, these results suggest that colorectal patients undergoing tumor removal surgery may benefit from also receiving interferon-alpha through continuous delivery.

IFN-γ-dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis.

Tumor-associated lymphatic vessels promote metastasis and regulate antitumor immune responses. Here, we assessed the impact of cytotoxic T cells on the local lymphatic vasculature and concomitant tumor dissemination during an antitumor response. Interferon-γ (IFN-γ) released by effector T cells enhanced the expression of immunosuppressive markers by tumor-associated lymphatic endothelial cells (LECs). However, at higher effector T cell densities within the tumor, T cell-based immunotherapies induced LEC apoptosis and decreased tumor lymphatic vessel density. As a consequence, lymphatic flow was impaired, and lymph node metastasis was reduced. Mechanistically, T cell-mediated tumor cell death induced the release of tumor antigens and cross-presentation by tumor LECs, resulting in antigen-specific LEC killing by T cells. When LECs lacked the IFN-γ receptor expression, LEC killing was abrogated, indicating that IFN-γ is indispensable for reducing tumor-associated lymphatic vessel density and drainage. This study provides insight into how cytotoxic T cells modulate tumor lymphatic vessels and may help to improve immunotherapeutic protocols.

Gliadin-reactive vitamin D-sensitive proinflammatory ILCPs are enriched in celiac patients.

Celiac disease (CD) is a multisystem disease in which different organs may be affected. We investigate whether circulating innate lymphoid cells (ILCs) contribute to the CD peripheral inflammatory status. We find that the CD cytokine profile is characterized by high concentrations of IL-12p40, IL-18, and IFN-γ, paralleled by an expansion of ILC precursors (ILCPs). In the presence of the gliadin peptides p31-43 and pα-9, ILCPs from CD patients increase transglutaminase 2 (TG2) expression, produce IL-18 and IFN-γ, and stimulate CD4+ T lymphocytes. IFN-γ is also produced upon stimulation with IL-12p40 and IL-18 and is inhibited by the addition of vitamin D. Low levels of blood vitamin D correlate with high IFN-γ and ILCP presence and mark the CD population mostly affected by extraintestinal symptoms. Dietary vitamin D supplementation appears to be an interesting therapeutic approach to dampen ILCP-mediated IFN-γ production.

Food-Grade Titanium Dioxide Induces Toxicity in the Nematode Caenorhabditis elegans and Acute Hepatic and Pulmonary Responses in Mice.

Food-grade titanium dioxide (E171) contains variable percentages of titanium dioxide (TiO2) nanoparticles (NPs), posing concerns for its potential effects on human and animal health. Despite many studies, the actual relationship between the physicochemical properties of E171 NPs and their interaction with biological targets is still far from clear. We evaluated the impact of acute E171 administration on invertebrate and vertebrate animals. In the nematode, Caenorhabditis elegans, the administration of up to 1.0 mg/mL of E171 did not affect the worm's viability and lifespan, but significantly impaired its pharyngeal function, reproduction, and development. We also investigated whether the intravenous administration of E171 in mice (at the dose of 6 mg/kg/body weight) could result in an acute over-absorption of filter organs. A significant increase of hepatic titanium concentration and the formation of microgranulomas were observed. Interstitial inflammation and parenchymal modification were found in the lungs, coupled with titanium accumulation. This was probably due to the propensity of TiO2 NPs to agglomerate, as demonstrated by transmission electron microscopy experiments showing that the incubation of E171 with serum promoted the formation of compact clusters. Overall, these data emphasize the actual risk for human and animal exposure to E171.

Phylogeny and morphology of Ophiocordyceps puluongensis sp. nov. (Ophiocordycipitaceae, Hypocreales), a new fungal pathogen on termites from Vietnam.

Termites are serious pests in agriculture and forestry, causing significant economic losses to property and the construction industry. However, only a few entomopathogenic fungi attack termites that are dominant members of most terrestrial biomes. This study contributes to the taxonomic knowledge of entomopathogenic fungi with the description of a new pathogen on termites collected from the Pu Luong Nature Reserve in Vietnam. The new termite pathogen, Ophiocordyceps puluongensis, is introduced on the basis of morphological and multigene phylogenetic evidence. Based on the combined dataset of five genes including the nuclear ribosomal small and large subunits (nrSSU and nrLSU), the elongation factor 1α (tef-1α), and the largest and the second largest subunits of RNA polymerase II (rpb1 and rpb2), phylogenetic analyses were performed by Maximum Likelihood and Bayesian inference methods to determine the phylogenetic position of O. puluongensis. Three samples of O. puluongensis are clustered in the Hirsutella thompsonii subclade of Hirsutella lineages in Ophiocordyceps, and clustered together with O. asiatica to form a separate clade from other Ophiocordyceps species. Morphologically, O. puluongensis differs from O. asiatica by its smaller and shorter perithecia, asci and ascospores, pink to reddish-orange stipes of stromata, as well as smaller fusiform or citriform conidia. The distinctiveness of this termite pathogen is strongly supported by both molecular phylogeny and morphology. The entomopathogenic fungus O. puluongensis could have the potential to be used as bioinsecticides to control termites.

CD4+c-Met+Itgα4+ T cell subset promotes murine neuroinflammation.

OBJECTIVE: c-Met, a tyrosine kinase receptor, is the unique receptor for hepatocyte growth factor (HGF). The HGF/c-Met axis is reported to modulate cell migration, maturation, cytokine production, and antigen presentation. Here, we report that CD4+c-Met+ T cells are detected at increased levels in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). METHODS: c-Met expression by CD4+ T cells was analyzed mostly by flow cytometry and by immunohistochemistry from mice and human PBMCs. The in vivo role of CD4+c-Met+ T cells was assessed in EAE. RESULTS: CD4+c-Met+ T cells found in the CNS during EAE peak disease are characterized by a pro-inflammatory phenotype skewed towards a Th1 and Th17 polarization, with enhanced adhesion and transmigration capacities correlating with increased expression of integrin α4 (Itgα4). The adoptive transfer of Itgα4-expressing CD4+Vα3.2+c-Met+ T cells induces increased disease severity compared to CD4+Vα3.2+c-Met- T cells. Finally, CD4+c-Met+ T cells are detected in the brain of MS patients, as well as in the blood with a higher level of Itgα4. These results highlight c-Met as an immune marker of highly pathogenic pro-inflammatory and pro-migratory CD4+ T lymphocytes associated with neuroinflammation.

Multigene phylogeny and morphology reveal Ophiocordycepshydrangea sp. nov. and Ophiocordycepsbidoupensis sp. nov. (Ophiocordycipitaceae).

Ophiocordyceps species have a wide range of insect hosts, from solitary beetle larva to social insects. However, among the species of Ophiocordyceps, only a few attack cicada nymphs. These species are mainly clustered in the Ophiocordycepssobolifera clade in Ophiocordyceps. A new entomopathogenic fungus parasitic on cicada nymphs, and another fungus parasitic on the larva of Coleoptera, are described in this study. The two new species viz. Ophiocordycepshydrangea and Ophiocordycepsbidoupensis were introduced based on morphology and multigene phylogenetic evidence. The phylogenetic framework of Ophiocordyceps was reconstructed using a multigene (nrSSU, nr LSU, tef-1α, rpb1, and rpb2) dataset. The phylogenetic analyses results showed that O.hydrangea and O.bidoupensis were statistically well-supported in the O.sobolifera clade, forming two separate subclades from other species of Ophiocordyceps. The distinctiveness of these two new species was strongly supported by both molecular phylogeny and morphology.

Repeated administration of the food additive E171 to mice results in accumulation in intestine and liver and promotes an inflammatory status.

Titanium dioxide (TiO2) is widely used in pharmaceuticals preparations, cosmetics, and as a food additive (E171). It contains microparticles and a fraction of nanoparticles (NPs) which can be absorbed systemically by humans after ingestion. Increasing concern has been aroused about the impact of oral exposure to TiO2 NPs from dietary and non-dietary sources on human health. In spite of several toxicological studies conducted in recent years, a solid risk assessment of oral exposure to E171 has not been satisfactorily achieved. We investigated whether repeated oral administration of E171 to mice at a dose level (5 mg/kg body weight for 3 days/week for 3 weeks) comparable to estimated human dietary exposure, results in TiO2 deposition in the digestive system and internal organs, and in molecular and cellular alterations associated with an inflammatory response. To reproduce the first phase of digestion, a new administration approach involving the dripping of the E171 suspension into the mouth of mice was applied. Significant accumulation of titanium was observed in the liver and intestine of E171-fed mice; in the latter a threefold increase in the number of TiO2 particles was also measured. Titanium accumulation in liver was associated with necroinflammatory foci containing tissue monocytes/macrophages. Three days after the last dose, increased superoxide production and inflammation were observed in the stomach and intestine. Overall, the present study indicates that the risk for human health associated with dietary exposure to E171 needs to be carefully considered.

Adaptive Shape Ripening and Interparticle Bridging of l-Arginine-Stabilized Silica Nanoparticles during Evaporative Colloidal Crystal Assembly.

During evaporative self-assembly of colloidal crystal films, spherical l-arginine-stabilized silica colloids adapt to different close-packed geometries by faceting and forming bridge connections with their nearest neighbors. We systematically studied the morphological changes of 37 and 138 nm diameter colloids during evaporative assembly and compared them to 65 nm Stöber silica colloids prepared without l-arginine. Colloidal crystal films were grown from particles that had been dialyzed against water or l-arginine, and tetraethyl orthosilicate (TEOS) and/or l-arginine were added to solutions during colloidal film growth. Solid-state 29Si NMR spectra showed the presence of l-arginine and incompletely condensed silica in colloids grown from silica seeds in l-arginine solutions. These colloids were especially susceptible to chemical ripening during the colloidal assembly process, adopting faceted shapes that reflected the packing symmetry of the colloidal crystal films. The addition of l-arginine and TEOS accelerated these shape changes by catalyzing the hydrolysis and olation of silica and by adding a source of silica to the solution, respectively. This chemistry provides a route to single-component and binary colloidal crystals composed of nonspherical silica building blocks.

High mobility group box 1 orchestrates tissue regeneration via CXCR4.

Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.

Binary Colloidal Crystal Films Grown by Vertical Evaporation of Silica Nanoparticle Suspensions.

Despite intensive research efforts in the synthesis of binary colloidal crystals, the production of well ordered binary colloidal crystal films over large areas continues to be synthetically challenging. In this paper, we investigate the phase behavior of binary mixtures of l-arginine-stabilized 36 and 22 nm silica nanoparticles deposited as centimeter-scale thin films onto a vertical substrate via evaporative assembly. By adjusting the temperature and relative colloid composition under high humidity conditions, we controlled the order of the resultant colloidal crystal films. The domain size of the AB2 binary crystalline phase increased with an excess of small (B) particles and a very slow evaporation rate below 45 °C, with the best results obtained at 30° and 35 °C.

TACI-dependent APRIL signaling maintains autoreactive B cells in a mouse model of systemic lupus erythematosus.

Autoantibodies contribute to the development of systemic lupus erythematosus (SLE). APRIL (a proliferation-inducing ligand), a member of the TNF superfamily, regulates plasma-cell survival and binds to TACI (transmembrane activator CAML interactor) and BCMA (B-cell maturation antigen). We previously showed that APRIL blockade delayed disease onset in lupus-prone mice. In order to evaluate the role of APRIL receptors in the development of SLE, APRIL, TACI, BCMA, or double TACI.BCMA null mutations were introduced into the Nba2.Yaa (Y-linked autoimmune acceleration) spontaneous lupus mouse model. Mortality as a consequence of glomerulonephritis (GN) was reduced in Nba2.APRIL-/- .Yaa, Nba2.TACI-/- .Yaa and double-KO mice compared with Nba2.Yaa mice and correlated with lower levels of circulating antibodies, while splenic populations remained unchanged. In contrast, the appearance of symptoms was accelerated in BCMA-deficient mice, in which TACI signaling was increased. Finally, lupus-prone mice deficient for the APRIL-TACI axis produced less pathogenic antibodies and developed less GN. Disease reduction was attributed to impaired T-independent type 2 responses when the APRIL-TACI signaling axis was disrupted. Collectively, our results have identified and confirmed APRIL as a new target involved in B-cell activation, in the maintenance of plasma cell survival and subsequent increased autoantibody production that sustains lupus development in mice.

IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment.

Colorectal cancer (CRC) metastatic dissemination to the liver is one of the most life-threatening malignancies in humans and represents the leading cause of CRC-related mortality. Herein, we adopted a gene transfer strategy into mouse hematopoietic stem/progenitor cells to generate immune-competent mice in which TEMs-a subset of Tie2(+) monocytes/macrophages found at peritumoral sites-express interferon-alpha (IFNα), a pleiotropic cytokine with anti-tumor effects. Utilizing this strategy in mouse models of CRC liver metastasis, we show that TEMs accumulate in the proximity of hepatic metastatic areas and that TEM-mediated delivery of IFNα inhibits tumor growth when administered prior to metastasis challenge as well as on established hepatic lesions, improving overall survival. Further analyses unveiled that local delivery of IFNα does not inhibit homing but limits the early phases of hepatic CRC cell expansion by acting on the radio-resistant hepatic microenvironment. TEM-mediated IFNα expression was not associated with systemic side effects, hematopoietic toxicity, or inability to respond to a virus challenge. Along with the notion that TEMs were detected in the proximity of CRC metastases in human livers, these results raise the possibility to employ similar gene/cell therapies as tumor site-specific drug-delivery strategies in patients with CRC.

Toll-like receptor 8 deletion accelerates autoimmunity in a mouse model of lupus through a Toll-like receptor 7-dependent mechanism.

Systemic lupus erythematosus is an autoimmune disorder characterized by increased levels of lymphocyte activation, antigen presentation by dendritic cells, and the formation of autoantibodies. This leads to immune complex-mediated glomerulonephritis. Toll-like receptor 7 (T7) and TLR9 localize to the endosomal compartment and play important roles in the generation of autoantibodies against nuclear components, as they recognize RNA and DNA, respectively. In contrast, very little is known about endogenous TLR8 activation in mice. We therefore tested whether TLR8 could affect autoimmune responses in a murine model of lupus. We introduced a Tlr8 null mutation into C57BL/6 mice congenic for the Nba2 (NZB autoimmunity 2) locus and bearing the Yaa (Y-linked autoimmune acceleration) mutation containing a tlr8 duplicated gene, and monitored disease development, autoantibody production, and glomerulonephritis-associated mortality. Cellular responses were investigated in female Nba2.TLR8(-/-) mice bearing no copy of tlr8. The TLR8 deficiency accelerated disease progression and mortality, increased the number of circulating antibodies and activated monocytes, and heightened cellular responses to TLR7 ligation. TLR8-deficient antigen-presenting cells exhibited increased levels of MHC class II expression. The ability of dendritic cells to present antigens to allogeneic T cells after TLR7 ligation was also improved by TLR8 deficiency. TLR8 deletion accelerated autoimmunity in lupus-prone mice in response to TLR7 activation. Antigen-presenting cell function seemed to play a key role in mediating the effects of TLR8 deficiency.

O-linked glycosylation determines the nephritogenic potential of IgA rheumatoid factor.

Deficient glycosylation of O-linked glycans in the IgA1 hinge region is associated with IgA nephropathy in humans, but the pathogenic contribution of the underlying structural aberrations remains incompletely understood. We previously showed that mice implanted with cells secreting the class-switch variant 6-19 IgA anti-IgG2a rheumatoid factor, but not 46-42 IgA anti-IgG2a rheumatoid factor, develop glomerular lesions resembling IgA nephropathy. Because the levels of O-linked glycosylation in the hinge region and the structures of N-linked glycans in the CH1 domain differ in 6-19 IgA and 46-42 IgA, we determined the respective contributions of O- and N-linked glycans to the nephritogenic potential of the 6-19 IgA rheumatoid factor in mice. Wild-type 6-19 IgA secreted by implanted cells induced significant formation of glomerular lesions, whereas poorly O-glycosylated 6-19 IgA glycovariants or a 6-19 IgA hinge mutant lacking O-linked glycans did not. However, we observed no apparent heterogeneity in the structure of N-linked glycans attached to three different sites of the Fc regions of nephritogenic and non-nephritogenic 6-19 IgAs. Collectively, our data suggest a critical role of O-linked glycans attached to the hinge region in the development of IgA nephropathy-like GN induced by 6-19 IgA rheumatoid factor in mice.

TLR7 signaling exacerbates CNS autoimmunity through downregulation of Foxp3+ Treg cells.

The innate Toll-like receptor 7 (TLR7) detects infections by recognizing viral and bacterial single-stranded RNA. In addition to pathogen-derived RNA, immune cells expressing high levels of TLR7, such as B cells and dendritic cells (DCs), can be activated by self-RNA. During myelin-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, TLR7 expression is increased within the central nervous system (CNS). To define the contribution of TLR7 to the development of EAE, we evaluated the course of the disease in C57BL/6-Tlr7-deficient mice compared with that in WT mice and found that TLR7-deficient mice had decreased disease severity. This protection was associated with decreased myelin oligodendrocyte glycoprotein-specific T-cell activation by primed DCs, decreased circulating autoantibodies, attenuated inflammation within the CNS, and increased Foxp3(+) regulatory T cells in the periphery and in the CNS. In conclusion, we show that TLR7 is involved in the maintenance of autoimmunity in the pathogenesis of EAE.