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.

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.

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.

The Neutrophil: Constant Defender and First Responder.

The role of polymorphonuclear neutrophils (PMNs) in biology is often recognized during pathogenesis associated with PMN hyper- or hypo-functionality in various disease states. However, in the vast majority of cases, PMNs contribute to resilience and tissue homeostasis, with continuous PMN-mediated actions required for the maintenance of health, particularly in mucosal tissues. PMNs are extraordinarily well-adapted to respond to and diminish the damaging effects of a vast repertoire of infectious agents and injurious processes that are encountered throughout life. The commensal biofilm, a symbiotic polymicrobial ecosystem that lines the mucosal surfaces, is the first line of defense against pathogenic strains that might otherwise dominate, and is therefore of critical importance for health. PMNs regularly interact with the commensal flora at the mucosal tissues in health and limit their growth without developing an overt inflammatory reaction to them. These PMNs exhibit what is called a para-inflammatory phenotype, and have reduced inflammatory output. When biofilm growth and makeup are disrupted (i.e., dysbiosis), clinical symptoms associated with acute and chronic inflammatory responses to these changes may include pain, erythema and swelling. However, in most cases, these responses indicate that the immune system is functioning properly to re-establish homeostasis and protect the status quo. Defects in this healthy everyday function occur as a result of PMN subversion by pathological microbial strains, genetic defects or crosstalk with other chronic inflammatory conditions, including cancer and rheumatic disease, and this can provide some avenues for therapeutic targeting of PMN function. In other cases, targeting PMN functions could worsen the disease state. Certain PMN-mediated responses to pathogens, for example Neutrophil Extracellular Traps (NETs), might lead to undesirable symptoms such as pain or swelling and tissue damage/fibrosis. Despite collateral damage, these PMN responses limit pathogen dissemination and more severe damage that would otherwise occur. New data suggests the existence of unique PMN subsets, commonly associated with functional diversification in response to particular inflammatory challenges. PMN-directed therapeutic approaches depend on a greater understanding of this diversity. Here we outline the current understanding of PMNs in health and disease, with an emphasis on the positive manifestations of tissue and organ-protective PMN-mediated inflammation.

Periodontitis is an inflammatory disease of oxidative stress: We should treat it that way.

Periodontitis is a highly prevalent disease. As it progresses, it causes serious morbidity in the form of periodontal abscesses and tooth loss and, in the latter stages, pain. It is also now known that periodontitis is strongly associated with several nonoral diseases. Thus, patients with periodontitis are at greater risk for the development and/or exacerbation of diabetes, chronic obstructive pulmonary disease, and cardiovascular diseases, among other conditions. Although it is without question that specific groups of oral bacteria which populate dental plaque play a causative role in the development of periodontitis, it is now thought that once this disease has been triggered, other factors play an equal, and possibly more important, role in its progression, particularly in severe cases or in cases that prove difficult to treat. In this regard, we allude to the host response, specifically the notion that the host, once infected with oral periodontal pathogenic bacteria, will mount a defense response mediated largely through the innate immune system. The most abundant cell type of the innate immune system - polymorphonuclear neutrophils - can, when protecting the host from microbial invasion, mount a response that includes upregulation of proinflammatory cytokines, matrix metalloproteinases, and reactive oxygen species, all of which then contribute to the tissue damage and loss of teeth commonly associated with periodontitis. Of the mechanisms referred to here, we suggest that upregulation of reactive oxygen species might play one of the most important roles in the establishment and progression of periodontitis (as well as in other diseases of inflammation) through the development of oxidative stress. In this overview, we discuss both innate and epigenetic factors (eg, diabetes, smoking) that lead to the development of oxidative stress. This oxidative stress then provides an environment conducive to the destructive processes observed in periodontitis. Therefore, we shall describe some of the fundamental characteristics of oxidative stress and its effects on the periodontium, discuss the diseases and other factors that cause oxidative stress, and, finally, review potentially novel therapeutic approaches for the management (and possibly even the reversal) of periodontitis, which rely on the use of therapies, such as resveratrol and other antioxidants, that provide increased antioxidant activity in the host.

Differential response of human blood leukocytes to brushite, monetite, and calcium polyphosphate biomaterials.

Calcium phosphate-based biomaterials are extensively used for bone replacement and regeneration in orthopedic, dental, and maxillofacial surgical applications. The injury induced by surgical implantation of bone replacement graft materials initiates a cascade of host responses, starting with blood-biomaterial contact, protein adsorption on the material surface, blood coagulation, and leukocyte responses. During the initial acute inflammatory response, polymorphonuclear neutrophils (PMNs) and monocytes, abundant circulating leukocytes of the myeloid lineage, are recruited to the site of inflammation. In addition to responding to pathogenic challenges, these cells respond to particulate substances within the body including crystals of monosodium urate (MSU). Host responses toward grafts impact short- and long-term success in tissue engineering and regenerative applications. Although multinucleated osteoclasts, formed by monocyte/macrophage fusion, are generally thought to be responsible for resorption of implant biomaterials, the ability of different biomaterials to trigger PMNs, which are invariably present at the early stages after implant surgery, and are abundant in the oral cavity, has never been tested. In this article, we present analysis of the response of human blood-derived PMNs and monocytes toward brushite, monetite, and calcium polyphosphate (CPP) biomaterial substrates and compare this to the response to MSU crystals, the latter serving as a positive control. Employing multicolor flow cytometry to look at PMN and monocyte cell surface markers of activation to gauge the response to different biomaterials, we found that both types of myeloid cells are highly activated after exposure to brushite, monetite, and MSU but not CPP. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:253-262, 2020.

The Crossroads of Periodontitis and Oral Squamous Cell Carcinoma: Immune Implications and Tumor Promoting Capacities.

Periodontitis (PD) is increasingly considered to interact with and promote a number of inflammatory diseases, including cancer. In the case of oral squamous cell carcinoma (OSCC) the local inflammatory response associated with PD is capable of triggering altered cellular events that can promote cancer cell invasion and proliferation of existing primary oral carcinomas as well as supporting the seeding of metastatic tumor cells into the gingival tissue giving rise to secondary tumors. Both the immune and stromal components of the periodontium exhibit phenotypic alterations and functional differences during PD that result in a microenvironment that favors cancer progression. The inflammatory milieu in PD is ideal for cancer cell seeding, migration, proliferation and immune escape. Understanding the interactions governing this attenuated anti-tumor immune response is vital to unveil unexplored preventive or therapeutic possibilities. Here we review the many commonalities between the oral-inflammatory microenvironment in PD and oral-inflammatory responses that are associated with OSCC progression, and how these conditions can act to promote and sustain the hallmarks of cancer.

Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance.

Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoattraction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.

Lack of p47(phox) in Akita Diabetic Mice Is Associated with Interstitial Pneumonia, Fibrosis, and Oral Inflammation.

Excess reactive oxygen species production is central to the development of diabetic complications. The contribution of leukocyte reactive oxygen species produced by the NADPH oxidase to altered inflammatory responses associated with uncontrolled hyperglycemia is poorly understood. To get insight into the role of phagocytic superoxide in the onset of diabetic complications, we used a model of periodontitis in mice with chronic hyperglycemia and lack of leukocyte p47(phox) (Akita/Ncf1) bred from C57BL/6-Ins2(Akita)/J (Akita) and neutrophil cytosolic factor 1 knockout (Ncf1) mice. Akita/Nfc1 mice showed progressive cachexia starting at early age and increased mortality by six months. Their lungs developed infiltrative interstitial lesions that obliterated air spaces as early as 12 weeks when fungal colonization of lungs also was observed. Neutrophils of Akita/Ncf1 mice had normal degranulation and phagocytic efficiency when compared with wild-type mice. Although Akita/Ncf1 mice had increased prevalence of oral infections and more severe periodontitis compared with wild-type mice, bone loss was only marginally higher compared with Akita and Ncf1 null mice. Altogether these results indicate that lack of leukocyte superoxide production in mice with chronic hyperglycemia results in interstitial pneumonia and increased susceptibility to infections.

The 3BP2 adapter protein is required for chemoattractant-mediated neutrophil activation.

3BP2 is a pleckstrin homology and Src homology 2 domain-containing adapter protein mutated in cherubism, a rare autosomal-dominant human bone disorder. Previously, we have demonstrated a functional role for 3BP2 in peripheral B cell development and in peritoneal B1 and splenic marginal zone B cell-mediated Ab responses. In this study, we show that 3BP2 is required for G protein-coupled receptor-mediated neutrophil functions. Neutrophils derived from 3BP2-deficient (Sh3bp2-/-) mice failed to polarize their actin cytoskeleton or migrate in response to a gradient of chemotactic peptide, fMLF. Sh3bp2-/- neutrophils failed to adhere, crawl, and emigrate out of the vasculature in response to fMLF superfusion. 3BP2 is required for optimal activation of Src family kinases, small GTPase Rac2, neutrophil superoxide anion production, and for Listeria monocytogenes bacterial clearance in vivo. The functional defects observed in Sh3bp2-/- neutrophils may partially be explained by the failure to fully activate Vav1 guanine nucleotide exchange factor and properly localize P-Rex1 guanine nucleotide exchange factor at the leading edge of migrating cells. Our results reveal an obligate requirement for the adapter protein 3BP2 in G protein-coupled receptor-mediated neutrophil function.

Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition.

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.

The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma.

On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.