Inflammatory Response Mechanisms of the Dentine-Pulp Complex and the Periapical Tissues

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation

Non-invasive characterization of hybrid gelatin:poly-L-lactide electrospun scaffolds using second harmonic generation and multiphoton imaging

In this work we generated hybrid gelatin:poly-l-lactide electrospun scaffolds and implemented non-invasive methods to characterize them

Methods of Isolation and Analysis of TREG Immune Infiltrates from Injured and Dystrophic Skeletal Muscle

The immune infiltrate present in acutely injured or dystrophic skeletal muscle has been shown to play an important role in the process of muscle regeneration. Our work has described, for the first time, muscle regulatory T cells (Tregs), a unique population in phenotype and function capable of promoting skeletal muscle repair. Herein, we describe the methods we have optimized to study muscle Tregs, including their isolation from injured muscle, immuno-labeling for analysis/separation by flow cytometry, and measurement of their proliferation status

Genetic determinants of co-accessible chromatin regions in activated T cells across humans.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression

Pro-Inflammatory CD11c+CD206+ Adipose Tissue Macrophages Are Associated With Insulin Resistance in Human Obesity

OBJECTIVE: Insulin resistance and other features of the metabolic syndrome have been causally linked to adipose tissue macrophages (ATMs) in mice with diet-induced obesity. We aimed to characterize macrophage phenotype and function in human subcutaneous and omental adipose tissue in relation to insulin resistance in obesity. RESEARCH DESIGN AND METHODS: Adipose tissue was obtained from lean and obese women undergoing bariatric surgery. Metabolic markers were measured in fasting serum and ATMs characterized by immunohistology, flow cytometry, and tissue culture studies. RESULTS ATMs comprised CD11c(+)CD206(+) cells in "crown" aggregates and solitary CD11c(-)CD206(+) cells at adipocyte junctions. In obese women, CD11c(+) ATM density was greater in subcutaneous than omental adipose tissue and correlated with markers of insulin resistance. CD11c(+) ATMs were distinguished by high expression of integrins and antigen presentation molecules; interleukin (IL)-1beta, -6, -8, and -10; tumor necrosis factor-alpha; and CC chemokine ligand-3, indicative of an activated, proinflammatory state. In addition, CD11c(+) ATMs were enriched for mitochondria and for RNA transcripts encoding mitochondrial, proteasomal, and lysosomal proteins, fatty acid metabolism enzymes, and T-cell chemoattractants, whereas CD11c(-) ATMs were enriched for transcripts involved in tissue maintenance and repair. Tissue culture medium conditioned by CD11c(+) ATMs, but not CD11c(-) ATMs or other stromovascular cells, impaired insulin-stimulated glucose uptake by human adipocytes. CONCLUSIONS: These findings identify proinflammatory CD11c(+) ATMs as markers of insulin resistance in human obesity. In addition, the machinery of CD11c(+) ATMs indicates they metabolize lipid and may initiate adaptive immune responses

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These finding provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously

One naive T cell, multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets

Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8(+ )T cells by dendritic cells loaded with killed allogeneic breast cancer cells

INTRODUCTION: The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity. METHODS: In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8(+ )T cells in 2-week and/or 3-week cultures. CD8(+ )T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens. RESULTS: We found that DCs loaded with killed breast cancer cells can prime naïve CD8(+ )T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201(- )breast cancer cells can kill HLA-A*0201(+ )breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin. CONCLUSION: This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer

Phenotypic and Functional Properties of Helios+ Regulatory T Cells

Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios+ and Helios− Treg, we profiled cell-surface markers of FoxP3+ Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios+ Treg and that a Helios+ Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios+ Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios+ Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios+ Treg proliferated more than Helios− Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios+ cells in culture. Taken together, these data show that Helios+ Treg represent a functional subset with associated CD103 and GITR expression