A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Myeloperoxidase Gene Deletion Causes Drastic Microbiome Shifts in Mice and Does Not Mitigate Dextran Sodium Sulphate-Induced Colitis.

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

NK cell profiling in West Nile virus encephalitis reveals potential metabolic basis for functional inhibition.

Natural killer (NK) cells are cytotoxic lymphocytes important for viral defense. West Nile virus (WNV) infection of the central nervous system (CNS) causes marked recruitment of bone marrow (BM)-derived monocytes, T cells and NK cells, resulting in severe neuroinflammation and brain damage. Despite substantial numbers of NK cells in the CNS, their function and phenotype remain largely unexplored. Here, we demonstrate that NK cells mature from the BM to the brain, upregulate inhibitory receptors and show reduced cytokine production and degranulation, likely due to the increased expression of the inhibitory NK cell molecule, MHC-I. Intriguingly, this correlated with a reduction in metabolism associated with cytotoxicity in brain-infiltrating NK cells. Importantly, the degranulation and killing capability were restored in NK cells isolated from WNV-infected tissue, suggesting that WNV-induced NK cell inhibition occurs in the CNS. Overall, this work identifies a potential link between MHC-I inhibition of NK cells and metabolic reduction of their cytotoxicity during infection.

Dysbiosis in imiquimod-induced psoriasis alters gut immunity and exacerbates colitis development.

Psoriasis has long been associated with inflammatory bowel disease (IBD); however, a causal link is yet to be established. Here, we demonstrate that imiquimod-induced psoriasis (IMQ-pso) in mice disrupts gut homeostasis, characterized by increased proportions of colonic CX3CR1hi macrophages, altered cytokine production, and bacterial dysbiosis. Gut microbiota from these mice produce higher levels of succinate, which induce de novo proliferation of CX3CR1hi macrophages ex vivo, while disrupted gut homeostasis primes IMQ-pso mice for more severe colitis with dextran sulfate sodium (DSS) challenge. These results demonstrate that changes in the gut environment in psoriasis lead to greater susceptibility to IBD in mice, suggesting a two-hit requirement, that is, psoriasis-induced altered gut homeostasis and a secondary environmental challenge. This may explain the increased prevalence of IBD in patients with psoriasis.

Dietary protein increases T-cell-independent sIgA production through changes in gut microbiota-derived extracellular vesicles.

Secretory IgA is a key mucosal component ensuring host-microbiota mutualism. Here we use nutritional geometry modelling in mice fed 10 different macronutrient-defined, isocaloric diets, and identify dietary protein as the major driver of secretory IgA production. Protein-driven secretory IgA induction is not mediated by T-cell-dependent pathways or changes in gut microbiota composition. Instead, the microbiota of high protein fed mice produces significantly higher quantities of extracellular vesicles, compared to those of mice fed high-carbohydrate or high-fat diets. These extracellular vesicles activate Toll-like receptor 4 to increase the epithelial expression of IgA-inducing cytokine, APRIL, B cell chemokine, CCL28, and the IgA transporter, PIGR. We show that succinate, produced in high concentrations by microbiota of high protein fed animals, increases generation of reactive oxygen species by bacteria, which in turn promotes extracellular vesicles production. Here we establish a link between dietary macronutrient composition, gut microbial extracellular vesicles release and host secretory IgA response.

Impact of Dietary Fiber on West Nile Virus Infection.

Dietary fiber supports healthy gut bacteria and their production of short-chain fatty acids (SCFA), which promote anti-inflammatory cell development, in particular, regulatory T cells. It is thus beneficial in many diseases, including influenza infection. While disruption of the gut microbiota by antibiotic treatment aggravates West Nile Virus (WNV) disease, whether dietary fiber is beneficial is unknown. WNV is a widely-distributed neurotropic flavivirus that recruits inflammatory monocytes into the brain, causing life-threatening encephalitis. To investigate the impact of dietary fiber on WNV encephalitis, mice were fed on diets deficient or enriched with dietary fiber for two weeks prior to inoculation with WNV. To induce encephalitis, mice were inoculated intranasally with WNV and maintained on these diets. Despite increased fecal SCFA acetate and changes in gut microbiota composition, dietary fiber did not affect clinical scores, leukocyte infiltration into the brain, or survival. After the brain, highest virus loads were measured in the colon in neurons of the submucosal and myenteric plexuses. Associated with this, there was disrupted gut homeostasis, with shorter colon length and higher local inflammatory cytokine levels, which were not affected by dietary fiber. Thus, fiber supplementation is not effective in WNV encephalitis.

Dietary carbohydrate, particularly glucose, drives B cell lymphopoiesis and function.

While diet modulates immunity, its impact on B cell ontogeny remains unclear. Using mixture modeling, a large-scale isocaloric dietary cohort mouse study identified carbohydrate as a major driver of B cell development and function. Increasing dietary carbohydrate increased B cell proportions in spleen, mesenteric lymph node and Peyer's patches, and increased antigen-specific immunoglobulin G production after immunization. This was linked to increased B lymphopoiesis in the bone marrow. Glucose promoted early B lymphopoiesis and higher total B lymphocyte numbers than fructose. It drove B cell development through glycolysis and oxidative phosphorylation, independently of fatty acid oxidation in vitro and reduced B cell apoptosis in early development via mTOR activation, independently of interleukin-7. Ours is the first comprehensive study showing the impact of macronutrients on B cell development and function. It shows the quantitative and qualitative interplay between dietary carbohydrate and B cells and argues for dietary modulation in B cell-targeting strategies.

Targeting the gut to treat multiple sclerosis.

The gut-brain axis (GBA) refers to the complex interactions between the gut microbiota and the nervous, immune, and endocrine systems, together linking brain and gut functions. Perturbations of the GBA have been reported in people with multiple sclerosis (pwMS), suggesting a possible role in disease pathogenesis and making it a potential therapeutic target. While research in the area is still in its infancy, a number of studies revealed that pwMS are more likely to exhibit altered microbiota, altered levels of short chain fatty acids and secondary bile products, and increased intestinal permeability. However, specific microbes and metabolites identified across studies and cohorts vary greatly. Small clinical and preclinical trials in pwMS and mouse models, in which microbial composition was manipulated through the use of antibiotics, fecal microbiota transplantation, and probiotic supplements, have provided promising outcomes in preventing CNS inflammation. However, results are not always consistent, and large-scale randomized controlled trials are lacking. Herein, we give an overview of how the GBA could contribute to MS pathogenesis, examine the different approaches tested to modulate the GBA, and discuss how they may impact neuroinflammation and demyelination in the CNS.

How Changes in the Nutritional Landscape Shape Gut Immunometabolism.

Cell survival, proliferation and function are energy-demanding processes, fuelled by different metabolic pathways. Immune cells like any other cells will adapt their energy production to their function with specific metabolic pathways characteristic of resting, inflammatory or anti-inflammatory cells. This concept of immunometabolism is revolutionising the field of immunology, opening the gates for novel therapeutic approaches aimed at altering immune responses through immune metabolic manipulations. The first part of this review will give an extensive overview on the metabolic pathways used by immune cells. Diet is a major source of energy, providing substrates to fuel these different metabolic pathways. Protein, lipid and carbohydrate composition as well as food additives can thus shape the immune response particularly in the gut, the first immune point of contact with food antigens and gastrointestinal tract pathogens. How diet composition might affect gut immunometabolism and its impact on diseases will also be discussed. Finally, the food ingested by the host is also a source of energy for the micro-organisms inhabiting the gut lumen particularly in the colon. The by-products released through the processing of specific nutrients by gut bacteria also influence immune cell activity and differentiation. How bacterial metabolites influence gut immunometabolism will be covered in the third part of this review. This notion of immunometabolism and immune function is recent and a deeper understanding of how lifestyle might influence gut immunometabolism is key to prevent or treat diseases.

Immune Modulation of Monocytes Dampens the IL-17+ γδ T Cell Response and Associated Psoriasis Pathology in Mice.

Psoriasis is a chronic inflammatory autoimmune skin condition that affects millions of people worldwide. It is driven by IL-17-producing CD4 and γδ T cells and targeted by current anti-IL-17 or anti-IL-23 mAb therapies. These treatments are expensive, increase the risk of opportunistic infections, and do not specifically target the inflammatory cascade. Other cells, including inflammatory monocytes, have been shown to migrate to psoriatic plaques in both human disease and the imiquimod-induced mouse model and could thus constitute potential alternative therapeutic targets. In the mouse, immune modifying particles (IMPs) specifically target Ly6Chi inflammatory monocytes migrating to the site of inflammation, sequestering them in the spleen. In this project, we determined whether IMPs could mitigate the development of imiquimod -induced psoriasis in mice. IMP treatment significantly reduced imiquimod-induced psoriasis severity, decreasing dermal infiltration of Ly6Chi monocytes as well as early-stage monocyte-derived dermal macrophages. This was associated with reduced levels of hallmark cytokines IL-23 and IL-1ß as well as associated IL-17-producing γδ T cells. Our work highlights the crucial importance of inflammatory monocytes in the development of this disease as well as a therapeutic potential for IMP in psoriasis.

The protein corona determines the cytotoxicity of nanodiamonds: implications of corona formation and its remodelling on nanodiamond applications in biomedical imaging and drug delivery.

The use of nanodiamonds for biomedical and consumer applications is growing rapidly. As their use becomes more widespread, so too do concerns around their cytotoxicity. The cytotoxicity of nanodiamonds correlates with their cellular internalisation and circulation time in the body. Both internalisation and circulation time are influenced by the formation of a protein corona on the nanodiamond surface. However, a precise understanding of both how the corona forms and evolves and its influence on cytotoxicity is lacking. Here, we investigated protein corona formation and evolution in response to two classes of nanodiamonds, pristine and aminated, and two types of proteins, bovine serum albumin and fibronectin. Specifically, we found that a corona made of bovine serum albumin (BSA), which represents the most abundant protein in blood plasma, reduced nanodiamond agglomeration. Fibronectin (FN9-10), the second most abundant protein found in the plasma, exhibited a significantly higher nanodiamond binding affinity than BSA, irrespective of the nanodiamond surface charge. Finally, nanodiamonds with a BSA corona displayed less cytotoxicity towards nonphagocytic liver cells. However, regardless of the type of corona (FN9-10 or BSA), both classes of nanodiamonds induced substantial phagocytic cell death. Our results emphasise that a precise understanding of the corona composition is fundamental to determining the fate of nanoparticles in the body.

Corrigendum: Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction.

[This corrects the article DOI: 10.3389/fnut.2019.00057.].

Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction.

The interaction between gut microbiota and host plays a central role in health. Dysbiosis, detrimental changes in gut microbiota and inflammation have been reported in non-communicable diseases. While diet has a profound impact on gut microbiota composition and function, the role of food additives such as titanium dioxide (TiO2), prevalent in processed food, is less established. In this project, we investigated the impact of food grade TiO2 on gut microbiota of mice when orally administered via drinking water. While TiO2 had minimal impact on the composition of the microbiota in the small intestine and colon, we found that TiO2 treatment could alter the release of bacterial metabolites in vivo and affect the spatial distribution of commensal bacteria in vitro by promoting biofilm formation. We also found reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO2 significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8+ T cells, increased macrophages as well as increased expression of inflammatory cytokines. These findings collectively show that TiO2 is not inert, but rather impairs gut homeostasis which may in turn prime the host for disease development.

Detrimental Impact of Microbiota-Accessible Carbohydrate-Deprived Diet on Gut and Immune Homeostasis: An Overview.

Dietary fibers are non-digestible polysaccharides functionally known as microbiota-accessible carbohydrates (MACs), present in inadequate amounts in the Western diet. MACs are a main source of energy for gut bacteria so the abundance and variety of MACs can modulate gut microbial composition and function. This, in turn, impacts host immunity and health. In preclinical studies, MAC-deprived diet and disruption of gut homeostasis aggravate the development of inflammatory diseases, such as allergies, infections, and autoimmune diseases. The present review provides a synopsis on the impact of a low-MAC diet on gut homeostasis or, more specifically, on gut microbiota, gut epithelium, and immune cells.

The majority of murine γδ T cells at the maternal-fetal interface in pregnancy produce IL-17.

Compared with lymphoid tissues, the immune cell compartment at mucosal sites is enriched with T cells bearing the γδ T-cell receptor (TCR). The female reproductive tract, along with the placenta and uterine decidua during pregnancy, are populated by γδ T cells predominantly expressing the invariant Vγ6(+)Vδ1(+) receptor. Surprisingly little is understood about the function of these cells. We found that the majority of γδ T cells in the non-pregnant uterus, pregnant uterus, decidua and placenta of mice express the transcription factor RORγt and produce interleukin-17 (IL-17). In contrast, IFNγ-producing γδ T cells were markedly reduced in gestational tissues compared with uterine-draining lymph nodes and spleen. Both uterine-resident invariant Vγ6(+) and Vγ4(+) γδ T cells which are more typically found in lymphoid tissues and circulating blood, were found to express IL-17. Vγ4(+) γδ T cells were particularly enriched in the placenta, suggesting a pregnancy-specific recruitment or expansion of these cells. A small increase in IL-17-producing γδ T cells was observed in allogeneic compared with syngeneic pregnancy, suggesting a contribution to regulating the maternal response to paternally-derived alloantigens. However, their high proportions also in non-pregnant uteri and gestational tissues of syngeneic pregnancy imply a role in the prevention of intrauterine infection or quality control of fetal development. These data suggest the need for a more rigorous evaluation of the role of IL-17 in sustaining normal pregnancy, particularly as emerging data points to a pathogenic role for IL-17 in pre-eclampsia, pre-term birth, miscarriage and maternal immune activation-induced behavioral abnormalities in offspring.

Differential Responsiveness of Innate-like IL-17- and IFN-γ-Producing γδ T Cells to Homeostatic Cytokines.

γδ T cells respond to molecules upregulated following infection or cellular stress using both TCR and non-TCR molecules. The importance of innate signals versus TCR ligation varies greatly. Both innate-like IL-17-producing γδ T (γδT-17) and IFN-γ-producing γδ T (γδT-IFNγ) subsets tune the sensitivity of their TCR following thymic development, allowing robust responses to inflammatory cytokines in the periphery. The remaining conventional γδ T cells retain high TCR responsiveness. We determined homeostatic mechanisms that govern these various subsets in the peripheral lymphoid tissues. We found that, although innate-like γδT-17 and γδT-IFNγ cells share elements of thymic development, they diverge when it comes to homeostasis. Both exhibit acute sensitivity to cytokines compared with conventional γδ T cells, but they do not monopolize the same cytokine. γδT-17 cells rely exclusively on IL-7 for turnover and survival, aligning them with NKT17 cells; IL-7 ligation triggers proliferation, as well as promotes survival, upregulating Bcl-2 and Bcl-xL. γδT-IFNγ cells instead depend heavily on IL-15. They display traits analogous to memory CD8(+) T cells and upregulate Bcl-xL and Mcl-1 upon cytokine stimulation. The conventional γδ T cells display low sensitivity to cytokine-alone stimulation and favor IL-7 for their turnover, characteristics reminiscent of naive αß T cells, suggesting that they may also require tonic TCR signaling for population maintenance. These survival constraints suggest that γδ T cell subsets do not directly compete with each other for cytokines, but instead fall into resource niches with other functionally similar lymphocytes.