Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

A Primed Neutrophil Subset Predicts the Risk of Bloodstream Infections in Allogeneic Hematopoietic Stem-Cell Transplant Patients: A Prospective Study.

BACKGROUND: Bloodstream infections (BSIs) are the most common infectious complication in patients who receive allogeneic hematopoietic stem-cell transplants (allo-HSCTs). Polymorphonuclear neutrophils (PMNs) are quantified to monitor the susceptibility to BSIs; however, their degree of activation is not. We previously identified a population of primed PMNs (pPMNs) with distinct markers of activation representing approximately 10% of PMNs in circulation. In this study, we investigate whether susceptibility to BSIs is related to the proportion of pPMNs rather than strictly PMN counts. METHODS: In this prospective observational study, we used flow cytometry to assess pPMNs in blood and oral rinse samples collected from patients receiving an allo-HSCT over the course of their treatment. We used the proportion of pPMNs in the blood on day 5 post-transplant to categorize patients into a high- or a low-pPMN group (>10% or <10% pPMNs). These groups were then used as a predictor of BSIs. RESULTS: A total of 76 patients were enrolled in the study with 36 in the high-pPMN group and 40 in the low-pPMN group. Patients in the low-pPMN group had lower expression of PMN activation and recruitment markers and displayed a delay in PMN repopulation of the oral cavity after the transplant. These patients were more susceptible to BSIs compared with patients in the high-pPMN group with an odds ratio of 6.5 (95% confidence interval, 2.110-25.07; P = .002). CONCLUSIONS: In patients who receive an allo-HSCT, having <10% pPMNs early in the post-transplant phase can be an independent predictor of BSI in allo-HSCT patients.

Peptidomic Analysis of Urine from Youths with Early Type 1 Diabetes Reveals Novel Bioactivity of Uromodulin Peptides In Vitro.

Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis are captured by the urinary peptidome. To elucidate the early kidney response to chronic hyperglycemia, we conducted a peptidomic investigation into urines from otherwise healthy youths with type 1 diabetes and their non-diabetic peers using unbiased and targeted mass spectrometry-based techniques. This cross-sectional study included two separate cohorts for the discovery (n = 30) and internal validation (n = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. We further examined the subset of 162 peptides, which were quantified across all thirty samples. Of the 15 differentially excreted peptides (p < 0.05), seven derived from a C-terminal region (589SGSVIDQSRVLNLGPITRK607) of uromodulin, a kidney-specific protein. Increased excretion of five uromodulin peptides was replicated in the validation cohort using parallel reaction monitoring (p < 0.05). One of the validated peptides (SGSVIDQSRVLNLGPI) activated NFκB and AP-1 signaling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses highlighted several potential proteases such as hepsin, meprin A, and cathepsin B to be responsible for generating these peptides. In summary, we identified a urinary signature of uromodulin peptides associated with early type 1 diabetes before clinical manifestations of kidney disease and discovered novel bioactivity of uromodulin peptides in vitro Our present findings lay the groundwork for future studies to validate peptide excretion in larger and broader populations, to investigate the role of bioactive uromodulin peptides in high glucose conditions, and to examine proteases that cleave uromodulin.

The Effect of Intensity-Modulated Radiotherapy to the Head and Neck Region on the Oral Innate Immune Response and Oral Microbiome: A Prospective Cohort Study of Head and Neck Tumour Patients.

Neutrophils, also known as polymorphonuclear leukocytes (PMNs), form a significant component of the innate host response, and the consequence of the interaction between the oral microbiota and PMNs is a crucial determinant of oral health status. The impact of radiation therapy (RT) for head and neck tumour (HNT) treatment on the oral innate immune system, neutrophils in particular, and the oral microbiome has not been thoroughly investigated. Therefore, the objective of this study was to characterize RT-mediated changes in oral neutrophils (oPMNs) and the oral microbiome in patients undergoing RT to treat HNTs. Oral rinse samples were collected prior to, during and post-RT from HNT patients receiving RT at Dental Oncology at Princess Margaret Cancer Centre. The oPMNs counts and activation states were analysed using flow cytometry, and the oral microbiome was analysed using 16S rRNA gene sequencing. Statistically significant (p < 0.05) drops in oPMN counts and the activation states of the CD11b, CD16, CD18, CD64 and H3Cit markers from pre-RT to post-RT were observed. Moreover, exposure to RT caused a significant reduction in the relative abundance of commensal Gram-negative bacteria and increased the commensal Gram-positive microbes. Ionizing radiation for the treatment of HNTs simultaneously decreased the recruitment of oPMNs into the oral cavity and suppressed their activation state. The oral microbiome composition post-RT was altered significantly due to RT which may favour the colonization of specific microbial communities unfavourable for the long-term development of a balanced oral microbiome.

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria in vitro based on the surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap formation, and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.

Morphological characterization of para- and proinflammatory neutrophil phenotypes using transmission electron microscopy.

BACKGROUND AND OBJECTIVE: Bacterial challenge is constant in the oral cavity. To contain the commensal biofilm, partly activated neutrophils are continuously recruited as part of a normal physiologic process, without exposing the host to the harmful effect of a fully active neutrophil response. This intermediate immune state has been termed para-inflammation, as opposed to the fully activated proinflammatory state in oral disease. Directly visualizing these cells and their components via transmission electron microscopy (TEM) enhances our understanding of neutrophil activation state differences in oral health and disease, as obtained from molecular studies. The aim of this study was to describe the morphology of the para-inflammatory phenotype displayed by oral neutrophils in health, and compare it to the morphology of the naïve blood neutrophil, and the proinflammatory oral neutrophils in chronic periodontitis. This morphology was characterized by differences in granule content, phagosome content and cytoplasm and nuclear changes. We also examined the morphological changes induced in naïve neutrophils, which were stimulated in vitro by bacteria, and in oral neutrophils in full tissue samples in vivo. MATERIAL AND METHODS: Neutrophils were isolated from blood and saliva samples of patients with chronic periodontitis and healthy individuals. The cells were viewed under TEM and analyzed in imaging software examining granularity, cytoplasm density, euchromatin amount in the nucleus and phagosome content. A separate cohort of blood neutrophils was incubated with Streptococcus oralis and analyzed under TEM in the same manner. Gingival tissue samples were obtained from patients with chronic periodontitis and viewed under TEM, with the neutrophils present analyzed in the same manner. RESULTS: The proinflammatory cells showed less granulation, lighter cytoplasm and higher amount of nuclear euchromatin. These changes were accentuated in the proinflammatory oral chronic periodontitis neutrophils compared to the para-inflammatory oral health neutrophils. The oral chronic periodontitis neutrophils also contained more phagosomes and had more phagosomes containing undigested bacteria. These changes were partially reproduced in the naïve blood cells after exposing them to S. oralis. The neutrophils in the gingival tissues displayed naïve morphology when viewed in the blood vessels and gradually showed proinflammatory morphological changes as they traveled through the connective tissue into the epithelium. CONCLUSION: Oral neutrophils display morphological changes consistent with partial or full activation, corresponding to their para- or proinflammatory states. These changes can also be induced in naïve cells by incubating them with commensal bacteria. Neutrophils change their morphology towards an activated state as they travel through the gingival tissue.

Nuclear Factor Erythroid 2-Related Factor 2 Down-Regulation in Oral Neutrophils Is Associated with Periodontal Oxidative Damage and Severe Chronic Periodontitis.

The balance between reactive oxygen species and antioxidants plays an important role in periodontal health. We previously demonstrated that high reactive oxygen species production by oral polymorphonuclear neutrophils (oPMNs) in chronic periodontitis (CP) refractory to conventional therapy is associated with severe destruction of periodontium. Herein, we show that inhibition of antioxidant production through down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in oPMN, despite enhanced recruitment in the oral cavity, is associated with severe CP. Twenty-four genes in the Nrf2-mediated oxidative stress response pathway were down-regulated in PMNs of diseased patients. Downstream of Nrf2, levels of oPMN superoxide dismutase 1 and catalase were decreased in severe CP, despite increased recruitment. Nrf2(-/-) mice had more severe loss of periodontium in response to periodontitis-inducing subgingival ligatures compared with wild-types. Levels of 8-hydroxy-deoxyguanosine were increased in periodontal lesions of Nrf2(-/-) mice, indicating high oxidative damage. We report, for the first time, Nrf2 pathway down-regulation in oPMNs of patients with severe CP. PMNs of CP patients may be primed for low antioxidant response in the context of high recruitment in the oral cavity, resulting in increased oxidative tissue damage.

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry.

Neutrophils are the most abundant white blood cell and are an essential component of the innate immune system. A complete cataloguing of cell surface markers has not been undertaken for neutrophils isolated from circulation as well as healthy and inflamed tissues. To identify cell-surface markers specific to human neutrophils, we used high-throughput flow cytometry to screen neutrophil populations isolated from blood and oral rinses from healthy and chronic periodontitis patients against a panel of 374 known cluster of differentiation (CD) antibodies. This screen identified CD11b, CD16, and CD66b as markers that are consistently expressed on neutrophils independent of the cell location, level of activation and disease state. Cell sorting against CD11b, CD16 and CD66b allowed for the enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These findings suggest an ideal surface marker set for isolating mature neutrophils from humans. The screen also demonstrated that tissue neutrophils from chronically inflamed tissue display a unique surface marker set compared to tissue neutrophils present in healthy, non-inflamed tissues.

Rac-null leukocytes are associated with increased inflammation-mediated alveolar bone loss.

Periodontitis is characterized by altered host-biofilm interactions that result in irreversible inflammation-mediated alveolar bone loss. Genetic and epigenetic factors that predispose to ineffective control of biofilm composition and maintenance of tissue homeostasis are not fully understood. We elucidated how leukocytes affect the course of periodontitis in Rac-null mice. Mouse models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to loss of alveolar bone due to inflammation in Rac-null mice. Leukocyte margination was differentially impaired in these mice during attachment in conditional Rac1-null (granulocyte/monocyte lineage) mice and during rolling and attachment in Rac2-null (all blood cells) mice. Inflammatory responses to subgingival ligatures, assessed by changes in peripheral blood differential leukocyte numbers, were altered in Rac-null compared with wild-type mice. In response to persistent subgingival ligature-mediated challenge, Rac-null mice had increased loss of alveolar bone with patterns of resorption characteristic of aggressive forms of periodontitis. These findings were partially explained by higher osteoclastic coverage of the bone-periodontal ligament interface in Rac-null compared with wild-type mice. In conclusion, this study demonstrates that leukocyte defects, such as decreased endothelial margination and tissue recruitment, are rate-limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

Practical 10-Color T-Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide.

Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28Null T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28Null T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.

Rac regulates PtdInsP3 signaling and the chemotactic compass through a redox-mediated feedback loop.

Directional cell migration is an essential requirement for efficient neutrophil translocation to sites of infection and requires the establishment of a polarized cell characterized by an actin-rich leading edge facing the chemoattractant gradient. The asymmetrical accumulation of phosphatidylinositol(3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] in the up-gradient leading edge is a hallmark of polarization and regulates the recruitment and localization of various effector proteins at the leading-edge plasma membrane. How shallow gradients of chemoattractants trigger and maintain a much steeper intracellular gradient of PtdIns(3,4,5)P(3) is a critical question in the study of leukocyte chemotaxis. Our data demonstrate that the migration of neutrophils toward the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine depends on the generation of reactive oxygen species by the phagocytic NADPH oxidase (NOX2) and subsequent oxidation and inhibition of phosphatase and tensin homolog. Moreover, we show that events downstream of PtdIns(3,4,5)P(3), including phosphorylation of AKT, Rac activation, uncapping of actin filaments, and directional migration, can be attenuated by ROS scavengers or genetic ablation of NOX2. Using Rac mutants that are defective in their ability to activate NOX2, we show that Rac regulates a redox-mediated feedback loop that mediates directional migration of neutrophils.

Oral inflammatory load predicts vascular function in a young adult population: a pilot study.

Background: The periodontium is a highly vascularized area of the mouth, and periodontitis initiates negative functional and structural changes in the vasculature. However, mild oral inflammation, including levels experienced by many apparently healthy individuals, has an unclear impact on cardiovascular function. The purpose of this pilot study is to investigate the effects of objectively measured whole mouth oral inflammatory load (OIL) on vascular function in apparently healthy individuals. Methods: In this cross-sectional and correlational analysis, we recruited 28 young (18-30 years) and systemically healthy participants (16 male, 12 female). Using oral neutrophil counts, a validated measure for OIL, we collected participant's mouth rinse samples and quantified OIL. Blood pressure, arterial stiffness (pulse-wave velocity) and endothelial function (brachial artery flow-mediated dilation) were also measured. Results: Only oral neutrophil count significantly predicted flow-mediated dilation % (p = 0.04; R2 = 0.16, ß = - 1.05) and those with OIL levels associated with >2.5 × 105 neutrophil counts (n = 8) had a lower flow-mediated dilation % (6.0 ± 2.3%) than those with counts associated with gingival health with less than 2.5 × 105 neutrophil counts (10.0 ± 5.2%, p = 0.05). There were no significant predictors for arterial stiffness. Conclusion: We found that OIL was a predictor of reduced flow-mediated dilation. An impairment in flow-mediated dilation is an indicator of future possible risk of cardiovascular disease-one of the leading causes of death in North America. Therefore, this study provides evidence for the importance of oral health and that OIL may impact endothelial function.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Metronidazole enhances killing of Porphyromonas gingivalis by human PMNs.

Background and objectives: Periodontitis affects the supporting structures of the teeth as a result of the interactions between the subgingival biofilm and the host immune system. Periodontal therapy in severe forms of periodontitis often utilizes antimicrobial agents with some potential to improve host defense responses. In the present study, we investigated the in vitro effect of metronidazole (MTZ) at concentrations achievable in the periodontal pocket on PMN activation and PMN mediated killing of Porphyromonas gingivalis. Materials and methods: Flow cytometry based assays were used to measure the impact of MTZ on PMN degranulation, neutrophil extracellular trap (NET) formation and myeloperoxidase (MPO) release and phagocytosis in response to the keystone oral pathogen P. gingivalis. Functional assays for PMN mediated killing of P. gingivalis and reactive oxygen species (ROS) production in PMN were also carried out. Results: We demonstrate that PMNs pretreated with MTZ (2 µg/ml or 50 µg/ml) displayed enhanced killing of P. gingivalis compared to untreated PMNs. At concentrations achieved physiologically in the periodontal pocket, MTZ induced PMN surface expression of two activation markers (CD66 and CD63). MTZ did not alter P. gingivalis-induced NETosis, but suppressed P. gingivalis-induced ROS production and phagocytosis. Conclusion: MTZ displays a positive interaction with PMNs to potentiate PMN mediated killing of P. gingivalis and may therefore contribute to its beneficial effects in the treatment of periodontitis initiated by P. gingivalis infections including those refractory to conventional treatment.

The Association between Maternal Oral Inflammation and Neutrophil Phenotypes and Poly-Unsaturated Fatty Acids Composition in Human Milk: A Prospective Cohort Study.

This prospective cohort study aimed to investigate the impact of maternal oral inflammation on human milk composition including neutrophil counts, activation state (based on cluster of differentiation (CD) markers expression), and fatty acid levels. Fifty mothers were recruited from St. Michael's hospital, Toronto, and followed up from 2-4 weeks until 4 months postpartum. Oral rinse and human milk samples were collected at both timepoints. Oral polymorphonuclear neutrophils (oPMNs) within the rinses were quantified using flow cytometry and the participants' oral health state was categorized into three groups (i.e., healthy, moderate, and severe) based on the oPMNs counts. Fatty acids were identified and quantified using a gas chromatography-flame ionization detector (GC-FID). Compared to mothers with a healthy oral health state, mothers with moderate to severe oral inflammation had a statistically significant decrease in the expression of CD64 biomarker, an increase in the expression of CD14 biomarker on human milk neutrophils and a decrease in the levels of eicosapentaenoic acid (C20:5n-3) in their human milk at follow-up compared to baseline. This study demonstrates for the first time that maternal oral inflammation can affect human milk composition. The mechanism by which these alterations can affect infant health outcomes in the long term critically needs to be considered.

Neutrophil functions in patients with inherited bone marrow failure syndromes.

BACKGROUND: The inherited bone marrow failure syndromes (IBMFS) include Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome (SDS). Previous studies reported decreased neutrophil chemotaxis in patients with SDS; there are no reports of neutrophil function in other IBMFS. In this study we examined neutrophil respiratory burst function in IBMFS patients. PROCEDURE: Samples from 43 IBMFS patients and 61 healthy family members were collected, shipped, and analyzed within 24 hr. We also studied samples from 12 healthy control persons immediately after collection. Neutrophils were stimulated with phorbol 12-myristate acetate (PMA) and N-formyl-methyonyl-leucyl-phenylalanine (fMLP), and respiratory burst analyzed by reduction of dihydro-rhodamine and cytochrome c. RESULTS: There was no significant difference in the degree of fMLP or PMA-driven respiratory burst activity between each of the IBMFS subgroups and their respective family members. There was also no difference in respiratory burst activity between any IBMFS, pooled group of all healthy family members and healthy controls. CONCLUSIONS: Neutrophil respiratory burst activity from IBMFS patients does not differ from that of healthy family members and controls.

Human neutrophils coordinate chemotaxis by differential activation of Rac1 and Rac2.

Rac1 and Rac2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and Rac2-T17N fusion proteins to acutely alter the activity of Rac1 and Rac2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and Rac2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or Rac2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant.

The actin-binding protein Adseverin mediates neutrophil polarization and migration.

Through the process of chemotactic migration, neutrophils are able to recruit to an inflammatory site and eliminate pathogens, thus playing a vital role in host defense. The process of neutrophil chemotaxis is mediated by dynamic actin reorganization and polymerization. Adseverin, an actin-binding protein and member of the Gelsolin superfamily of proteins, has been hypothesized to regulate goal directed movement through the capping and severing of actin filaments, but has never been investigated in the context of neutrophil chemotaxis. Using an Adseverin knockout mouse model, we show that Adseverin plays a role in subcortical F-actin assembly at the leading edge during chemotaxis through the generation of free barbed ends on existing actin filaments. In addition, in the absence of Adseverin, neutrophils show reduced speed of migration in vitro and in vivo. This study indicates that Adseverin is a regulator of actin filament generation during neutrophil chemotaxis.

Design, Synthesis, Pharmacokinetics, and Biodistribution of a Series of Bone-Targeting EP4 Receptor Agonist Prodrugs for Treatment of Osteoporosis and Other Bone Conditions.

A series of bone-targeting EP4 receptor agonist conjugate prodrugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Singly and doubly radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream, and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up biodistribution and elimination studies. Doubly radiolabeled conjugate 4 was found to partition largely to the liver and bones, and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from bone-bound 4 with a half-time of about 7 days. When dosed orally, radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged, and only traces of radiolabeled 4 were found in the liver, blood, or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate, forming a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

Tissue-specific murine neutrophil activation states in health and inflammation.

Neutrophils are quickly recruited to tissues in response to proinflammatory cues; however, little is known about tissue neutrophil phenotypes in health. We employ a multicolor flow cytometric approach to assess surface markers of activation on neutrophils from the bone marrow, blood, peritoneum, spleen, liver, fat, colon, and oral cavity of healthy mice. Cell preparations were promptly fixed to preserve native surface marker expression levels. Peritoneal, colonic, and oral neutrophils were also assessed in the setting of pHrodo-induced peritonitis, dextran sodium sulfate-induced colitis, and ligature-induced periodontal disease, respectively. Our results demonstrate consistent detectable neutrophil populations in various sterile and nonsterile tissues of healthy mice, and these cells had tissue-specific neutrophil immunophenotypes. Neutrophils derived from biofilm-associated mucosal tissues had 2- to 3-fold higher expression of surface markers of activation, including CD66a, CD11b, and CD62L, compared to neutrophils derived from both sterile healthy tissues as well as tissues in animals treated with broad-spectrum antibiotics. Furthermore, the unique cluster of differentiation (CD) marker activation signatures of tissue-specific neutrophils from the peritoneum, colon, and oral cavity were altered to a proinflammatory immunophenotype with the presence of an inflammatory stimulus. Based on our results, we propose a model whereby a hierarchy of tissue neutrophil immunophenotypes, based on the differential expression of CD markers of activation, correlates with sterile, healthy commensal biofilm-associated and inflamed tissue states.