Primary Human Neutrophils Research Articles

High molecular pyrogens present in plant extracts interfere with examinations of their immunomodulatory properties in vitro

The widely accepted strategy to justify the use of medicinal plant extracts in diseases with inflammatory background is their examination on in vitro models using immune cells. It is also a key initial step of research for active principles, which could be then isolated and tested on more advanced models, becoming new pharmacologically active lead molecules. The crucial aspect which has not been so far addressed in this context, is the presence of pyrogens in plant preparations. The aim of this study was the examination of pyrogens interference with in vitro evaluation of anti-inflammatory activity of plant extracts using human primary neutrophils model together with introduction of effective method of interfering factors elimination. The obtained results showed that chosen plant extracts contained pyrogens, which were responsible for concentration-dependent stimulation of pro-inflammatory cytokines production by human neutrophils in vitro in the same extent as LPS did. The ultrafiltration method was successfully applied for pyrogens elimination, which effectiveness was confirmed using LAL test. The determined interference of pyrogens implies the necessity of their consideration and removal when in vitro studies include direct addition of plant extracts to the cell culture, what can be obtained by ultrafiltration, which does not affect extract composition.

Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity

Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.

Neutrophil activation by nanomaterials in vitro: comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses in vivo, a limited number of studies have investigated neutrophil responses to NMs in vitro. We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO2) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO2 NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to in vivo models where necessary.

Human neutrophils swim and phagocytise bacteria.

Leukocytes migrate in an amoeboid fashion while patrolling our organism in the search for infection or tissue damage. Their capacity to migrate has been proven integrin independent, however, non-specific adhesion or confinement remain a requisite in current models of cell migration. This idea has been challenged twice within the last decade with human neutrophils and effector T lymphocytes, which were shown to migrate in free suspension, a phenomenon termed swimming. While the relevance of leukocyte swimming in vivo remains under judgment, a growing amount of clinical evidence demonstrates that leukocytes are indeed found in liquid-filled body cavities, occasionally with phagocyted pathogens, such as in the amniotic fluid, the cerebrospinal fluid (CSF), or the eye vitreous and aqueous humor. We studied in vitro swimming of primary human neutrophils in the presence of live bacteria, in 2 and 3 dimensions. We show that swimming neutrophils perform phagocytosis of bacteria in suspension. By micropatterning live bacteria on a substrate with an optical technique, we further prove that they use chemotaxis to swim towards their targets. Moreover, we provide evidence that neutrophil navigation can alternate between adherent and non-adherent modes. Our results suggest that human neutrophils do not rely on adhesion to carry out their functions, supporting a versatile phagocytic function adaptable to the various environmental conditions encountered in vivo, as already suggested by clinical data. We verified a claim stated 10 years ago and never reproduced, on the capacity of human neutrophils to swim and perform swimming chemotaxis. We further extended those results to prove that swimming neutrophils can phagocytise bacteria, disregarding adhesion nor confinement as a requisite for accomplishing their function, which differs with current paradigms of leukocyte migration.

Recombinant Tissue Plasminogen Activator (r-tPA) Induces In-Vitro Human Neutrophil Migration via Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1).

Thrombolysis is the gold standard treatment for acute ischemic stroke. Besides its fibrinolytic role, recombinant tissue plasminogen activator (r-tPA) holds several non-fibrinolytic functions. Here, we investigated the potential role of r-tPA on human primary neutrophil migration in vitro. By means of modified Boyden chamber migration assay and checkerboard analysis we showed a dose-dependent chemotactic effect of r-TPA with a maximum effect reached by 0.03 mg/mL (0.003–1 mg/mL). Pre-incubation with MAP kinases inhibitors allowed the identification of PI3K/Akt, but not ERK1/2 as the intracellular pathway mediating the observed effects. Furthermore, by means of real-time PCR, immunocytochemistry and cytofluorimetry we demonstrated that the r-tPA receptor low density lipoprotein receptor-related protein 1 (LRP-1) is synthetized and expressed by neutrophils in response to r-tPA and TNF-α. Inhibition of LRP-1 by receptor-associated protein (RAP), prevented r-tPA-mediated F-actin polymerization, migration and signal through Akt but not ERK1/2. Lastly, also neutrophil degranulation in response to r-tPA seems to be mediated by LRP-1 under adhesion conditions. In conclusion, we show that r-tPA induces neutrophil chemotaxis through LRP-1/Akt pathway. Blunting r-tPA-mediated neutrophil activation might be beneficial as an adjuvant therapy to thrombolysis in this setting.

Leukocidins and the Nuclease Nuc Prevent Neutrophil-Mediated Killing of Staphylococcus aureus Biofilms.

Bacterial biofilms are linked with chronic infections and have properties distinct from those of planktonic, single-celled bacteria. The virulence mechanisms associated with Staphylococcus aureus biofilms are becoming better understood. Human neutrophils are critical for the innate immune response to S. aureus infection. Here, we describe two virulence strategies that converge to promote the ability of S. aureus biofilms to evade killing by neutrophils. Specifically, we show that while neutrophils exposed to S. aureus biofilms produce extracellular traps (NETs) and phagocytose bacteria, both mechanisms are inefficient in clearance of the biofilm biomass. This is attributed to the leukocidin LukAB, which promotes S. aureus survival during phagocytosis. We also show that the persistence of biofilm bacteria trapped in NETs is facilitated by S. aureus nuclease (Nuc)-mediated degradation of NET DNA. This study describes key aspects of the interaction between primary human neutrophils and S. aureus biofilms and provides insight into how S. aureus evades the neutrophil response to cause persistent infections.

Abstract 2842: Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor cells

Abstract During tumor development, neutrophils and other immune cells infiltrate the tumor niche. Neutrophils have been shown to have primarily pro-tumorigenic effects, and high levels of circulating neutrophils are indicative of a poor prognosis. Tumor-associated neutrophils (TANs) migrate to tumors by sensing chemical gradients produced by tumor cells; however, the mechanisms by which tumors and TANs interact and impact each other remain to be determined. It has been shown that malignant breast tumors, such as triple-negative breast tumors, harbor more TANs compared to less aggressive breast tumors. We hypothesize that neutrophil recruitment to the tumor niche depends on tumor-secreted, chemotactic factors that are primarily produced by malignant breast tumors. To test this, we used a panel of breast cancer cells grown in monolayers and more physiologically-relevant tumor spheroids, which were formed over the course of four days using the hanging-drop method. We characterized the size and morphology of the spheroids, which were generated from the poorly metastatic BT474 cells, as well as the highly malignant BT549 cells, MDA-MB-231 cells, and M4 cells of the MCF-10A cell series. We then compared the ability of tumor-conditioned media (TCM) isolated from either monolayers or spheroids to stimulate the migration of primary human neutrophils using transwell assays. We found that, compared to TCM isolated from control or poorly metastatic cell lines, the TCM derived from highly malignant cancer cells robustly stimulated the migration of neutrophils. These data indicate that breast cancer cells secrete factors with unique profiles that have varying potentials to recruit neutrophils. We are in the process of identifying specific tumor-secreted factors in the TCM using mass spectrometry. Additionally, we are assessing how the presence of neutrophils affects cancer cell migration and invasion by utilizing a coculture system in which tumor spheroids and primary neutrophils are imaged in real time. We reason that understanding the role of TANs during breast cancer progression will provide insight into novel therapeutic approaches, particularly in the context of highly metastatic forms of the disease. Citation Format: Lauren Hein, Shuvasree SenGupta, Yang Xu, Carole Parent. Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2842.

Structure-based design, semi-synthesis and anti-inflammatory activity of tocotrienolic amides as 5-lipoxygenase inhibitors

Inflammation contributes to the development of various pathologies, e.g. asthma, cardiovascular diseases, some types of cancer, and metabolic disorders. Leukotrienes (LT), biosynthesized from arachidonic acid by 5-lipoxygenase (5-LO), constitute a potent family of pro-inflammatory lipid mediators. δ-Garcinoic acid (δ-GA) (1), a natural vitamin E analogue, was chosen for further structural optimization as it selectively inhibited 5-LO activity in cell-free and cell-based assays without impairing the production of specialized pro-resolving mediators by 15-LO. A model of semi-quantitative prediction of 5-LO inhibitory potential developed during the current study allowed the design of 24 garcinamides that were semi-synthesized. In accordance with the prediction model, biological evaluations showed that eight compounds potently inhibited human recombinant 5-LO (IC50 < 100 nM). Interestingly, four compounds were substantially more potent than 1 in activated primary human neutrophils assays. Structure - activity relationships shed light on a supplementary hydrophobic pocket in the allosteric binding site that could be fitted with an aromatic ring.

Human primary neutrophil mRNA does not contaminate human resolving macrophage mRNA after efferocytosis

The ingestion of apoptotic corpses by macrophages, a process called efferocytosis, is a crucial step in inflammation resolution, since it alters macrophage phenotype toward a pro-resolving profile to foil inflammation and to favor tissue repair. Up to now, the resolving macrophages remain poorly characterized, especially in humans. Global investigations, like RNA sequencing, would be very helpful to unravel some features of these elusive cells. Nonetheless, these inquiries may be challenging in a single-species model, since the fate of ingested mRNA remains unknown and may hinder any subsequent mRNA investigations in the phagocyte. A full human model consisting of primary human neutrophil and primary human monocyte-derived macrophage co-culture was set up several decades ago to mimic in vitro the efferocytosis process. However, to our knowledge, this model has not been characterized as a suitable model to perform global mRNA investigations. Indeed, the extent of ingested neutrophil mRNA contamination has not been assessed in resolving macrophages. This work answers to this crucial question. Indeed, based on the protocols presented in this article, we demonstrate that neutrophil mRNA is severely degraded and is not able to cross-contaminate resolving macrophage mRNA, contrary to apoptotic human peripheral blood derived mononuclear cell (PBMC) or apoptotic leukemic Jurkat cell mRNA. Moreover, this allogenic co-culture system does not favor neither neutrophil activation nor macrophage pro-inflammatory cytokine release. Collectively, we highlight that this model of primary human neutrophil and primary human monocyte-derived macrophage co-culture is the best model for mRNA investigations in human resolving macrophages to help improving our knowledge on these crucial cells.

The Small GTPase Cdc42 Is a Major Regulator of Neutrophil Effector Functions.

Neutrophil granulocytes are key components of the innate immune system. As the first responders to inflammatory cues, they rapidly migrate toward the site of infection or inflammation and fulfill diverse effector functions. Since these effector functions can be both beneficial and harmful to the host and surrounding tissue, they require a strict control. The small GTPase Cdc42 is known to regulate neutrophil locomotion by controlling cytoskeleton rearrangement in murine neutrophils. However, the role of Cdc42 in other neutrophil functions in human neutrophils is still poorly understood. Here we demonstrate that in primary human neutrophils, Cdc42 controls directed and random migration, activation, and degranulation as well as the formation of reactive oxygen species, in a stimulus dependent manner. In addition, we show that Cdc42 regulates pathogen killing efficiency, both in murine and human neutrophils. Cdc42 regulation of neutrophil functions is linked to differential regulation of Akt, p38, and p42/44. Our data, therefore, suggests a mechanistic role for Cdc42 activity in primary human neutrophil biology, and identify Cdc42 activity as a target to modulate neutrophil effector mechanisms and killing efficacy.

An Attempt to Polarize Human Neutrophils Toward N1 and N2 Phenotypes in vitro.

Neutrophils act as the first line of defense against invading pathogens. Although traditionally considered in context of their antimicrobial effector functions, the importance of tumor-associated neutrophils (TANs) in the development of cancer has become increasingly clear during the last decade. With regard to their high plasticity, neutrophils were shown to acquire an anti-tumorigenic N1 or a pro-tumorigenic N2 phenotype. Despite the urgent need to get a comprehensive understanding of the interaction of TANs with their tumor microenvironment, most studies still rely on murine tumor models. Here we present for the first time a polarization attempt to generate N1 and N2 neutrophils from primary human neutrophils in vitro. Our results underscore that N1-polarized neutrophils have a pro-inflammatory phenotype characterized among others by a higher level of intercellular adhesion molecule (ICAM)-1 and high secretion of interferon (IFN)γ-induced protein 10 (IP-10)/C-X-C motif chemokine 10 (CXCL10) and tumor necrosis factor (TNF). Further, we demonstrate that neutrophils incubated under a tumor-mimicking in vitro environment show a high cell surface expression of C-X-C motif chemokine receptor 2 (CXCR2) and secrete high levels of interleukin (IL)-8. These findings suggest that it is feasible to polarize blood-derived primary human neutrophils toward N1- and N2-like phenotypes in vitro. Further, we hypothesized that the presence of anti-inflammatory neutrophil phenotype is not a phenomenon limited to cancer but also occurs when neutrophils are infected with intracellular pathogens. Indeed, our findings indicate that N2-polarized neutrophils exert a markedly decreased capacity to kill the protozoan parasite Leishmania donovani and therefore permit parasite persistence.

Targeted Amino Acid Substitution Overcomes Scale-Up Challenges with the Human C5a-Derived Decapeptide Immunostimulant EP67.

EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.

E‐Cigarette Use Increases Susceptibility to Bacterial Infection by Impairment of Human Neutrophil Chemotaxis, Phagocytosis and NET Formation

Although E‐cigarettes are frequently portrayed as a safer alternative to conventional cigarettes, a growing body of evidence suggests that the use of these devices may adversely affect host defense against infection. Neutrophils, critical effector cells of the innate immune system and early responders to sites of infection, may represent a particularly important cellular target of E‐cigarette vapor components. Using primary human neutrophils isolated from healthy donors, we evaluated the impact of electronic vapor extract (EVE) exposure on multiple aspects of neutrophil cellular physiology, including chemotaxis, reactive oxygen species (ROS) production, generation of Neutrophil Extracellular Traps (NETs) and phagocytosis. Using a transwell chamber approach, we found that exposure to E‐cigarette vapor‐infused media (EVE) reduced neutrophil chemotaxis in response to the chemoattractant f‐Met‐Leu‐Phe (fMLP) by up to 4.2 fold (p &lt; 0.001). Staining with the fluorescent actin probe rhodamine phalloidin revealed aberrant actin cytoskeleton polarization in EVE‐treated, fMLP‐stimulated neutrophils. Neutrophil membrane fluidity was also altered by EVE exposure, with holotomographic microscopy revealing alterations in cellular morphology in EVE‐treated neutrophils. ROS production was reduced in EVE‐treated neutrophils, and ROS‐dependent production of bactericidal extracellular traps was reduced by up to 3.5 fold (p &lt; 0.01). Interestingly, this reduction in NET production (NETosis) was observed in neutrophils treated with EVE produced using propylene glycol‐based E‐cigarette liquid formulations both with and without nicotine. The rate of neutrophil phagocytosis, quantified using fluorescent E. coli/S. aureus bioparticles, was also reduced by up to 47% in EVE‐treated neutrophils (p &lt; 0.05). To determine how such alterations of immune cell function might affect host response to infection, a physiologic mouse model of chronic E‐cigarette exposure and sepsis was utilized. In this model, E‐cigarette vapor inhalation led to reduced neutrophil migration into infected spaces and a higher burden of Pseudomonas aeruginosa. Taken together, these findings provide evidence that E‐cigarette use adversely impacts the innate immune system, potentially placing E‐cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.

Uric acid-mediated inflammasome activation in IL-6 primed innate immune cells is regulated by baricitinib

Objectives Gout is an inflammatory arthropathy caused by the deposition of monosodium urate (MSU). The synthesis and release of IL-1β is crucial for MSU-induced synovial inflammation. The aim of the present study was to investigate the mechanism of MSU crystal-induced autoinflammatory processes. Methods In vitro studies were used to evaluate the role of IL-6 in inflammasome activation in human neutrophils cultured with MSU crystals. Human neutrophils were stimulated with MSU in the presence or absence of IL-6 priming to determine NLRP3 inflammasome activation and subsequent cleaved caspase-1 induction or IL-1β production. Results IL-6 or MSU stimulation alone did not result in the efficient IL-1β production from human neutrophils. However, MSU stimulation induced marked IL-1β production from IL-6-primed neutrophils. Pretreatment with baricitinib, which blocks IL-6 receptor signaling, prevented MSU-induced cleaved caspase-1 or IL-1β induction in IL-6-primed neutrophils. Tocilizumab pretreatment also inhibited MSU-mediated IL-1β production from IL-6-primed neutrophils. Conclusion Priming of human neutrophils with IL-6 promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These results suggest that an endogenous cytokine, IL-6, is involved in MSU-mediated NLRP3 inflammasome activation and subsequent IL-1β production from innate immune cells and has a crucial role in MSU crystal-induced synovial inflammation. These findings provide insights into uric acid-mediated autoinflammation in the innate immune system.

Manuka honey modulates the release profile of a dHL-60 neutrophil model under anti-inflammatory stimulation

Manuka honey, a wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a current focus in the tissue engineering community as a tissue template additive. However, Manuka honey's effect on neutrophils during the inflammation-resolving phase has yet to be examined. This study investigates the effect of 0.5% and 3% Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes from a dHL-60 neutrophil model in the presence of anti-inflammatory stimuli (TGF-β, IL-4, IL-4 +IL-13). We hypothesized that Manuka honey would reduce the output of pro-inflammatory signals and increase the release of anti-inflammatory signals. The results of this study indicate that 0.5% honey significantly increases the release of CXCL8/IL-8, CCL2/MCP-1, CCL4/MIP-1β, CCL20/MIP-3α, IL-4, IL-1ra, and FGF-13 while reducing Proteinase 3 release in the anti-inflammatory-stimulated models. However, 3% honey significantly increased the release of TNF-α and CXCL8/IL-8 while reducing the release of all other analytes. We replicated a subset of the most notable findings in primary human neutrophils, and the consistent results indicate that the HL-60 data are relevant to the performance of primary cells. These findings demonstrate the variable effects of Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes of this model of neutrophil anti-inflammatory activity. This study reinforces the importance of tailoring the concentration of Manuka honey in a wound or tissue template to elicit the desired effects during the inflammation-resolving phase of wound healing. Future in vivo investigation should be undertaken to translate these results to a physiologically-relevant wound environment.

Response of Human Neutrophil Granulocytes to the Hyphae of the Emerging Fungal Pathogen Curvularia lunata.

Curvularia lunata is an ascomycete filamentous fungus causing local and invasive phaeohyphomycoses in both immunocompromised and immunocompetent patients. Neutrophils are crucial participants of the first line host defense against fungal infections. They migrate to the infected site and eliminate the infectious agents by various mechanisms including phagocytoses, oxidative damage, or formation of neutrophil extracellular trap (NET). Neutropenia may be a risk factor for phaeohyphomycoses, and restoration of the neutrophil function can improve the outcome of the infection. In the present study, interaction of primary human neutrophil granulocytes with the hyphae C. lunata was examined and compared to that with the well characterized filamentous fungal pathogen Aspergillus fumigatus. Neutrophils could recognize the serum opsonized hyphae of C. lunata and attach to them. Myeloperoxidase release was also activated by a soluble factor present in the culture supernatant of the fungus. Induction of the oxidative burst was found to depend on serum opsonization of the hyphae. Although extracellular hydrogen peroxide production was induced, the fungus efficiently blocked the oxidative burst by acidifying the reaction environment. This blockage also affected the NET forming ability of the neutrophils.

Suppression of neutrophilic inflammation can be modulated by the droplet size of anti-inflammatory nanoemulsions.

Aim: We aimed to develop nanoemulsions containing phosphodiesterase 4 inhibitor rolipram with different droplet sizes, to evaluate the anti-inflammatory effect against activated neutrophils and a related lung injury. Materials & methods: We prepared nanoemulsions of three different sizes, 68, 133 and 188nm. Results: The nanoemulsion inhibited the superoxide anion but not elastase release in primary human neutrophils. The large-sized nanoemulsions were mostly internalized by neutrophils, resulting in the reduction of intracellular Ca2+ half-life. The peripheral organ distribution of near-infrared dye-tagged nanoemulsions increased, following the decrease in droplet diameter. Rolipram entrapment into intravenous nanoemulsions ameliorated pulmonary inflammation. The smallest droplet size showed improvement, compared with the largest size. Conclusion: We established a foundation for the development of nanoemulsions against inflamed lung disease.

Human Neutrophil Granule Exocytosis in Response to Mycobacterium smegmatis.

Mycobacterium smegmatis rarely causes disease in the immunocompetent, but reported cases of soft tissue infection describe abscess formation requiring surgical debridement for resolution. Neutrophils are the first innate immune cells to accumulate at sites of bacterial infection, where reactive oxygen species and proteolytic enzymes are used to kill microbial invaders. As these phagocytic cells play central roles in protection from most bacteria, we assessed human neutrophil phagocytosis and granule exocytosis in response to serum opsonized or non-opsonized M. smegmatis mc2. Although phagocytosis was enhanced by serum opsonization, M. smegmatis did not induce exocytosis of secretory vesicles or azurophilic granules at any time point tested, with or without serum opsonization. At early time points, opsonized M. smegmatis induced significant gelatinase granule exocytosis compared to non-opsonized bacteria. Differences in granule release between opsonized and non-opsonized M. smegmatis decreased in magnitude over the time course examined, with bacteria also evoking specific granule exocytosis by six hours after addition to cultured primary single-donor human neutrophils. Supernatants from neutrophils challenged with opsonized M. smegmatis were able to digest gelatin, suggesting that complement and gelatinase granule exocytosis can contribute to neutrophil-mediated tissue damage seen in these rare soft tissue infections.

Inhibition of triggering receptor expressed on myeloid cells‐1 impairs thrombin generation

BackgroundNew evidence has shown the link between inflammation and thrombosis. Triggering receptor expressed on myeloid cells‐1 (TREM‐1) is an immunoreceptor expressed mostly on neutrophils and monocytes/macrophages. TREM‐1 acts as an amplifier of the inflammatory response, and its pharmacological inhibition displays protective effects in various models of inflammatory disorders, in particular by dampening coagulation abnormalities and thrombocytopenia observed during acute inflammation. ObjectivesWe aimed to decipher the role of TREM‐1 in fostering thrombin generation. MethodsWe measured thrombin generation (TG) by the use of calibrated automated thrombography with whole blood, and isolated primary human neutrophils and monocytes upon stimulation with lipopolysaccharide (LPS). Tissue factor (TF) expression was measured by flow cytometry and its activity by ELISA. Phosphatidylserine (PtdSer) exposure was determined by flow cytometry. A dodecapeptide (LR12) was used as a specific inhibitor of TREM‐1. ResultsLPS increased TG, TF expression, and activity, as well as the exposure of PtdSer on the surface of monocytes. LR12 dampened TF activity through the decrease of PtdSer exposure, leading to a reduction of thrombin generation. ConclusionsTREM‐1 inhibition decreases thrombin generation and could be an interesting target for the development of new inhibitors of leukocyte‐associated thrombotic activity.

Finegoldia magna, an Anaerobic Gram-Positive Bacterium of the Normal Human Microbiota, Induces Inflammation by Activating Neutrophils.

The Gram-positive anaerobic commensal Finegoldia magna colonizes the skin and other non-sterile body surfaces, and is an important opportunistic pathogen. Here we analyzed the effect of F. magna on human primary neutrophils. F. magna strains ALB8 (expressing protein FAF), 312 (expressing protein L) and 505 (naturally lacking both protein FAF and L) as well as their associated proteins activate neutrophils to release reactive oxygen species, an indication for neutrophil oxidative burst. Co-incubation of neutrophils with the bacteria leads to a strong increase of CD66b surface expression, another indicator for neutrophil activation. Furthermore, all tested stimuli triggered the release of NETs from the activated neutrophils, pointing to a host defense mechanism in response to the tested stimuli. This phenotype is dependent on actin rearrangement, NADPH oxidases and the ERK1/2 pathway. Proteins FAF and L also induced the secretion of several pro-inflammatory neutrophil proteins; HBP, IL-8 and INFγ. This study shows for the first time a direct interaction of F. magna with human neutrophils and suggests that the activation of neutrophils plays a role in F. magna pathogenesis.