Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils as well as of oral neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

TREM2 Promotes Immune Evasion by Mycobacterium tuberculosis in Human Macrophages.

Macrophage surface receptors are critical for pathogen defense, as they are the gatekeepers for pathogen entry and sensing, which trigger robust immune responses. TREM2 (triggering receptor expressed on myeloid cells 2) is a transmembrane surface receptor that mediates anti-inflammatory immune signaling. A recent study showed that TREM2 is a receptor for mycolic acids in the mycobacterial cell wall and inhibits macrophage activation. However, the underlying functional mechanism of how TREM2 regulates the macrophage antimycobacterial response remains unclear. Here, we show that Mycobacterium tuberculosis, the causative agent for tuberculosis, specifically binds to human TREM2 to disable the macrophage antibacterial response. Live but not killed mycobacteria specifically trigger the upregulation of TREM2 during macrophage infection through a mechanism dependent on STING (the stimulator of interferon genes). TREM2 facilitated uptake of M. tuberculosis into macrophages and is responsible for blocking the production of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and reactive oxygen species (ROS), while enhancing the production of interferon-ß (IFN-ß) and IL-10. TREM2-mediated blockade of ROS production promoted the survival of M. tuberculosis within infected macrophages. Consistent with this, genetic deletion or antibody-mediated neutralization of TREM2 reduced the intracellular survival of M. tuberculosis through enhanced production of ROS. Importantly, inhibition of type I IFN signaling in TREM2-overexpressing macrophages restored the ability of these cells to produce inflammatory cytokines and ROS, resulting in normal levels of intracellular bacteria killing. Collectively, our study identifies TREM2 as an attractive host receptor for host-directed antimycobacterial therapeutics. IMPORTANCE Mycobacterium tuberculosis is one of the most ancient bacterial pathogens and remains the leading cause of death from a single bacterial agent. The success of M. tuberculosis relies greatly on its ability to parasitize and disable its host macrophages. Previous studies have found that M. tuberculosis uses its unique cell wall lipids to manipulate the immune response by binding to specific surface receptors on macrophages. Our study reveals that M. tuberculosis binds to TREM2, an immunomodulatory receptor expressed on macrophages, to facilitate a "silent" mode of entry. Increased levels of TREM2 triggered by intracellular sensing of M. tuberculosis promoted the intracellular survival of M. tuberculosis through type I IFN-driven inhibition of reactive oxygen species (ROS) and proinflammatory cytokine production. Importantly, deletion of TREM2 reversed the effects of "silent" entry and resulted in increased production of inflammatory cytokines, generation of ROS, and cell death. As such, antibody-mediated or pharmacological targeting of TREM2 could be a promising strategy for novel treatments against M. tuberculosis infection.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Periodontal therapy increases neutrophil extracellular trap degradation.

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.

T Cell Proliferation Is Induced by Chronically TLR2-Stimulated Gingival Fibroblasts or Monocytes.

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly (p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1ß up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.

The Possible Role of Neutrophils in the Induction of Osteoclastogenesis.

The ligand of the receptor activator of NF-κB (RANKL) is a key molecule in the formation of osteoclasts, the key cells that cause the disease-associated alveolar bone resorption in periodontitis. We hypothesized that polymorphonuclear leukocytes (PMNs), found as the most prominent cells of inflamed periodontal tissues, could play an important role in providing signals to trigger osteoclastogenesis and thus activating pathological bone resorption in periodontitis. RANKL expression was investigated on circulatory PMNs (cPMNs) and oral PMNs (oPMNs) taken from both controls and periodontitis patients. On average, 2.3% and 2.4% RANKL expression was detected on the cPMNs and oPMNs from periodontitis patients, which did not differ significantly from healthy controls. Since cPMNs may acquire a more osteoclastogenesis-facilitating phenotype while migrating into the inflamed periodontium, we next investigated whether stimulated (with LPS, TNF-α, or IL-6) cPMNs have the capacity to contribute to osteoclastogenesis. Enduring surface expression of RANKL for short-lived cells as cPMNs was achieved by fixating stimulated cPMNs. RANKL expression on stimulated cPMNs, as assessed by flow cytometry and immunohistochemistry, was limited (6.48 ± 0.72%, mean expression ± SEM) after 24 and 48 hours of stimulation with LPS. Likewise, stimulation with TNF-α and IL-6 resulted in limited RANKL expression levels. These limited levels of expression did not induce osteoclastogenesis when cocultured with preosteoclasts for 10 days. We report that, under the aforementioned experimental conditions, neither cPMNs nor oPMNs directly induced osteoclastogenesis. Further elucidation of the key cellular players and immune mediators that stimulate alveolar bone resorption in periodontitis will help to unravel its pathogenesis.

The Challenge of Teaching Essential Immunology Laboratory Skills to Undergraduates in One Month-Experience of an Osteoimmunology Course on TLR Activation.

Acquiring immunology laboratory skills during undergraduate studies is often a prerequisite for admission to Masters' programs. Many broad liberal arts and sciences honors degree colleges struggle in teaching these essentials since only limited time is usually reserved for this. Here, we describe a new 1-month-course developed to train a small group of honors students in 6 techniques that are useful for immunology research. In essence, 15 students were divided into 3 groups of 5 students where each student became involved in current osteoimmunology research. Osteoimmunology is a relatively new branch of the immunology tree, where the effects of inflammation and the immune system on bone formation and bone degradation is studied. A broad, 3 weeks experiment on the chronic effects of molecules that specifically activate toll-like receptors TLR2 and TLR4 on bone formation or osteoclast differentiation was performed just before the start of the course. Control samples and samples treated with TLR2 (group A), TLR4 (group B), or TLR2+TLR4 (group C) agonists were harvested and analyzed using quantitative PCR, ELISA, biochemistry, microscopy of enzyme-histochemically stained osteoclasts, scanning electron microscopy, and confocal microscopy. Each technique was taught for 2 days by a specialized instructor, who was present at all laboratory activities. The primary research question for each group was: how does the experimental condition affect bone formation or osteoclast formation? The secondary research question specified per technique was: how does this technique answer part of the primary research question? Pedagogically, students were encouraged to collaborate within the group to analyze the obtained data. Secondly, at the end of the course, a representative of each group collaborated to summarize the TLR activation modalities of a technique of choice. Thirdly, each group wrote a report, where introduction and discussion were graded as a group; each technique part was graded individually. The summary of the results from the 3 treatment modalities was presented orally. The student evaluation of the course was high, students remarked that the course had a curriculum overarching function, since it created an awareness and appreciation for both the joy and the blood-sweat-and-tears aspects of pipetting, and writing research articles, making interpretation of those easier.

Oral Neutrophils Characterized: Chemotactic, Phagocytic, and Neutrophil Extracellular Trap (NET) Formation Properties.

Maintenance of oral health is in part managed by the immune-surveillance and antimicrobial functions of polymorphonuclear leukocytes (PMNs), which migrate from the circulatory system through the oral mucosal tissues as oral PMNs (oPMNs). In any microorganism-rich ecosystem, such as the oral cavity, PMNs migrate toward various exogenous chemoattractants, phagocytose bacteria, and produce neutrophil extracellular traps (NETs) to immobilize and eliminate pathogens. PMNs obtained from the circulation through venipuncture (hereafter called cPMNs) have been widely studied using various functional assays. We aimed to study the potential of oPMNs in maintaining oral health and therefore compared their chemotactic and antimicrobial functions with cPMNs. To establish chemotactic, phagocytic, and NET forming capacities, oPMNs and cPMNs were isolated from healthy subjects without obvious oral inflammation. Directional chemotaxis toward the chemoattractant fMLP was analyzed using an Insall chamber and video microscopy. fMLP expression was assessed by flow cytometry. Phagocytosis was analyzed by flow cytometry, following PMN incubation with heat-inactivated FITC-labeled micro-organisms. Furthermore, agar plate-based killing assays were performed with Escherichia coli (Ec). NET formation by oPMNs and cPMNs was quantified fluorimetrically using SYTOX™ Green, following stimulation with either PMA or RPMI medium (unstimulated control). In contrast to cPMNs, the chemotactic responses of oPMNs to fMLP did not differ from controls (mean velocity ± SEM of cPMNs: 0.79 ± 0.24; of oPMNs; 0.10 ± 0.07 micrometer/min). The impaired directional movement toward fMLP by oPMNs was explained by significantly lower fMLP receptor expression. Increased adhesion and internalization of various micro-organisms by oPMNs was observed. oPMNs formed 13 times more NETs than stimulated cPMNs, in both unstimulated and stimulated conditions. Compared to cPMNs, oPMNs showed a limited ability for intracellular killing of Ec. In conclusion, oPMNs showed exhausted capacity for efficient chemotaxis toward fMLP which may be the result of migration through the oral tissues into the oral cavity, being a highly "hostile" ecosystem. Overall, oPMNs' behavior is consistent with hyperactivity and frustrated killing. Nevertheless, oPMNs most likely contribute to maintaining a balanced oral ecosystem, as their ability to internalize microbes in conjunction with their abundant NET production remains after entering the oral cavity.

Survival, Retention, and Selective Proliferation of Lymphocytes Is Mediated by Gingival Fibroblasts.

Periodontitis, a chronic inflammatory disease of the periodontium, is characterized by osteoclast-mediated alveolar bone destruction. Gingival fibroblasts (GFs) present in the bone-lining mucosa have the capacity to activate the formation of osteoclasts, but little is known about which local immune cells (co-)mediate this process. The aim of this study was to investigate the cellular interactions of GFs with immune cells, including the contribution of GFs to osteoclast formation and their possible role in the proliferation of these immune cells. In addition, we investigated the expression of adhesion molecules and the inflammatory cytokines that are evoked by this interaction. GFs were cocultured with peripheral blood mononuclear cells (PBMCs), CD14+ monocytes or peripheral blood lymphocytes (PBLs) for 7, 14, and 21 days. After 21 days, comparable numbers of multinucleated cells (osteoclasts) were found in gingival fibroblast (GF)-PBMC and GF-monocyte cocultures. No osteoclasts were formed in GF-PBL cocultures, indicating that the PBLs present in GF-PBMC cocultures do not contribute to osteoclastogenesis. Persisting mononuclear cells were interacting with osteoclasts in GF-PBMC cocultures. Remarkably, a predominance of CD3+ T cells was immunohistochemically detected in GF cocultures with PBLs and PBMCs for 21 days that frequently interacted with osteoclasts. Significantly more T, B (CD19+), and NK (CD56+CD3-) cells were identified with multicolor flow cytometry in both GF-PBMC and GF-PBL cocultures compared to monocultures without GFs at all time points. GFs retained PBLs independently of the presence of monocytes or osteoclasts over time, showing a stable population of T, B, and NK cells between 7 and 21 days. T helper and cytotoxic T cell subsets remained stable over time in GF cocultures, while the number of Th17 cells fluctuated. Lymphocyte retention is likely mediated by lymphocyte-function-associated antigen-1 (LFA-1) expression, which was significantly higher in GF-PBL cultures compared to GF-monocyte cultures. When assessing inflammatory cytokine expression, high tumor necrosis alpha expression was only observed in the GF-PBMC cultures, indicating that this tripartite presence of GFs, monocytes, and lymphocytes is required for such an induction. Carboxyfluorescein succinimidyl ester-labeling showed that only the CD3+ cells proliferated in presence of GFs. This study demonstrates a novel role for GFs in the survival, retention, and selective proliferation of lymphocytes.

Titanium dioxide food additive (E171) induces ROS formation and genotoxicity: contribution of micro and nano-sized fractions.

Since 1969, the European Union approves food-grade titanium dioxide (TiO2), also known as E171 colouring food additive. E171 is a mixture of micro-sized particles (MPs) and nano-sized particles (NPs). Previous studies have indicated adverse effects of oral exposure to E171, i.e. facilitation of colon tumour growth. This could potentially be partially mediated by the capacity to induce reactive oxygen species (ROS). The aim of the present study is to determine whether E171 exposure induces ROS formation and DNA damage in an in vitro model using human Caco-2 and HCT116 cells and to investigate the contribution of the separate MPs and NPs TiO2 fractions to these effects. After suspension of the particles in Hanks' balanced salt solution buffer and cell culture medium with either bovine serum albumin (BSA) or foetal bovine serum, characterization of the particles was performed by dynamic light scattering, ROS formation was determined by electron spin/paramagnetic resonance spectroscopy and DNA damage was determined by the comet and micronucleus assays. The results showed that E171, MPs and NPs are stable in cell culture medium with 0.05% BSA. The capacity for ROS generation in a cell-free environment was highest for E171, followed by NPs and MPs. Only MPs were capable to induce ROS formation in exposed Caco-2 cells. E171, MPs and NPs all induced single-strand DNA breaks. Chromosome damage was shown to be induced by E171, as tested with the micronucleus assay in HCT116 cells. In conclusion, E171 has the capability to induce ROS formation in a cell-free environment and E171, MPs and NPs have genotoxic potential. The capacity of E171 to induce ROS formation and DNA damage raises concerns about potential adverse effects associated with E171 (TiO2) in food.